Genetically engineered gas vesicle gene clusters, genetic circuits, vectors, prokaryotic cells, compositions, methods and systems for contrast-enhanced imaging

ABSTRACT

Hybrid gas vesicle gene cluster (GVGC) configured for expression in a prokaryotic host are described comprising gas vesicle assembly (GVA) genes native to a GVA prokaryotic species and capable of being expressed in a functional form in the prokaryotic host, and one or more gas vesicle structural (GVS) genes native to one or more GVS prokaryotic species, at least one of the one or more GVS prokaryotic species different from the GVA prokaryotic species, and related gas vesicle reporting (GVR) genetic circuits, genetic, vectors, engineered cells, and related compositions methods and systems to produce GVs, hybrid GVGC and/or image a target site.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/413,206, entitled “Acoustic reporter genes for noninvasiveimaging of microbes in mammalian hosts” filed on Oct. 26, 2016, which isincorporated herein by reference in its entirety. The presentapplication also claims priority to U.S. Provisional Application No.62/367,750, entitled “Acoustomagnetic imaging with gas-filled proteinnanostructures” filed on Jul. 28, 2016, which is incorporated herein byreference in its entirety. The present application is also related toco-pending U.S. application Ser. No. 15/613,104, entitled “Gas-filledStructures and related Compositions, methods and systems to image atarget site” filed on Jun. 2, 2017, and to co-pending U.S. applicationSer. No. 15/663,600 entitled “Gas filled structure and relatedcompositions methods and systems for magnetic resonance imaging” filedon Jul. 28, 2017, each of which is also incorporated herein by referencein its entirety.

STATEMENT OF INTEREST

This invention was made with government support under Grant No. EB018975awarded by the National Institute of Health. The government has certainrights in the invention.

FIELD

The present disclosure relates to gas-filled structures, and inparticular genetically engineered gas vesicle gene clusters, relatedgenetic circuits, vectors, prokaryotic cells, compositions, methods andsystems to produce gas filled structures and/or to image biologicalevents in a target site, with particular reference to imaging performedby magnetic resonance imaging (MRI) and/or ultrasound.

BACKGROUND

Reporting biological events, such as a gene expression, proteolysis,biochemical reactions as well as cell location and function, iscurrently primarily based on fluorescent reporter genes.

Challenges remain for identifying, producing and/or developingbiocompatible reporters that can be imaged in deep tissues, enablemultiplexed imaging of biological events, are genetically modifiable,are capable of enabling detection at nanomolar concentrations and/orproduce dynamic contrast in response to local molecular signals.

SUMMARY

Provided herein are gas vesicle gene clusters (GVGC) that aregenetically engineered to include gas vesicle genes from at least twodifferent prokaryotic species to form a hybrid GVGC, and related gasvesicles (GVs), genetic circuits, vectors, genetically engineeredprokaryotic cells, compositions, methods and systems, which in severalembodiments can be used together with contrast-enhanced imagingtechniques such as such as magnetic resonance imaging (MRI) andultrasound, to detect and report biological events in an imaging targetsite.

According to a first aspect, a hybrid gas vesicle gene cluster (GVGC)configured for expression in a prokaryotic host is described. The hybridgene cluster comprises gas vesicle assembly (GVA) genes native to a GVAprokaryotic species and capable of being expressed in a functional formin the prokaryotic host. The hybrid gene cluster further comprises oneor more gas vesicle structural (GVS) genes native to one or more GVSprokaryotic species, at least one of the one or more GVS prokaryoticspecies different from the GVA prokaryotic species. In the hybrid genecluster the one or more gas vesicle structural genes and the gas vesicleassembly genes are in a configuration allowing co-expression of the gasvesicle structural genes and the gas vesicle assembly genes uponoperative connection with a regulatory sequence capable of operating inthe prokaryotic host. In some embodiments, the host is a prokaryote of asame prokaryotic species of the GVA prokaryotic species. In someembodiments, the host is a prokaryote of a prokaryotic species differentfrom the GVA prokaryotic species. In some embodiments, the GVAprokaryotic species is Bacillus Megaterium.

According to a second aspect a method is described to provide a hybridgas vesicle gene cluster (GVGC) configured for expression in aprokaryotic host and a hybrid gas vesicle gene cluster obtainablethereby. The method comprises: providing a polynucleotide constructcomprising gas vesicle assembly (GVA) genes native to a GVA prokaryoticspecies and capable of forming detectable GVs in the prokaryotic host.In the method, the polynucleotide construct further comprises gasvesicle structural (GVS) genes native to one or more GVS prokaryoticspecies, at least one of the one or more GVS prokaryotic speciesdifferent from the GVA prokaryotic species. In the polynucleotideconstruct, the GVA genes and the GVS genes are in a configurationallowing co-expression of the GVA genes and GVS genes upon operativeconnection of the GVA genes and GVS genes with a regulatory sequenceconfigured to operate in the prokaryotic host. In the method, theprokaryotic host is of a prokaryotic species different from the GVAprokaryotic species.

In some embodiments, the method further comprises detecting expressionin the prokaryotic host of one or more candidate GV gene clusters nativeto a prokaryotic species other than the prokaryotic host to obtain a GVAprokaryotic cell capable of forming detectable GVs in the prokaryotichost.

According to a third aspect, a method to produce a gas vesicle type in aprokaryotic host is described. The method comprises: introducing intothe prokaryotic host a hybrid gas vesicle gene cluster (GVGC) hereindescribed configured for expression in the prokaryotic host, in whichthe gas vesicle structural gene native to the second prokaryotic speciesencode for the gas vesicle type and, and expressing the hybrid GVGC inthe bacterial host to produce the gas vesicle type.

According to a fourth aspect, a method is described to image abiochemical event in a prokaryotic host comprised in an imaging targetsite, the method comprising:

introducing into the prokaryotic host a hybrid gas vesicle gene cluster(GVGC) herein described configured for expression in the prokaryotichost, the hybrid gas vesicle gene cluster (GVGC) encoding a gas vesicle(GV) type, wherein the GV type is a reportable molecular component of agas vesicle reporting (GVR) genetic circuit, in which molecularcomponents are connected one to another in accordance with a circuitdesign by activating, inhibiting, binding or converting reactions toform a fully connected network of interacting components, wherein in theGVR genetic circuit an expression of the GV type or an intracellularspatial translocation of the GV type occurs when the GVR genetic circuitoperates according to the circuit design in response to the biochemicalevent; and

imaging the target site comprising the prokaryotic host by applying amagnetic field and/or ultrasound to obtain an MRI and/or an ultrasoundimage of the target site.

The system comprises the hybrid GVGC, related GVR genetic circuits,related components and/or prokaryotic host cells in a combination forsimultaneous combined or sequential use in the imaging methods hereindescribed.

According to a fifth aspect, a method is described to label a targetprokaryotic host, the method comprising:

introducing into the target prokaryotic host a hybrid gas vesicle genecluster (GVGC) herein described configured for expression in the targetprokaryotic host, the hybrid gas vesicle gene cluster (GVGC) encoding agas vesicle (GV) type, wherein the GV type is a reportable molecularcomponent of a gas vesicle reporting (GVR) genetic circuit, in whichmolecular components are connected one to another in accordance with acircuit design by activating, inhibiting, binding or convertingreactions to form a fully connected network of interacting components,wherein in the GVR genetic circuit an expression of the GV type or anintracellular spatial translocation of the GV type occurs when the GVRgenetic circuit operates according to the circuit design in response toa trigger molecular component within the target prokaryotic host;

In the method, the introducing is performed under conditions resultingin presence of the trigger molecular component in the target prokaryotichost.

In some embodiments, the method can further comprise imaging the targetsite comprising the target prokaryotic host, by applying a magneticfield and/or ultrasound to obtain an MRI and/or a ultrasound image ofthe target site.

The system comprises the hybrid GVGC, related GVR genetic circuits,related components and/or prokaryotic host cells in a combination forsimultaneous combined or sequential use in the imaging methods hereindescribed.

According to a sixth aspect, a gas vesicle reporting (GVR) geneticcircuit is described, in which molecular components are connected one toanother in accordance with a circuit design by activating, inhibiting,binding or converting reactions to form a fully connected network ofinteracting components.

In the GVR genetic circuit, at least one reportable molecular componentis a hybrid GVGC herein described encoding a gas vesicle (GV) type, inwhich the gas vesicle (GV) type is expressed by the GVGC when thegenetic circuit operates according to the circuit design.

According to a seventh aspect, a vector is described comprising a hybridGVGC herein described configured for expression in a prokaryotic hostand/or one or more genetic molecular components of a Gas VesicleReporting (GVR) genetic circuit herein described configured to beoperated in the prokaryotic host. The vector is configured to introducethe hybrid GVGC, and/or one or more genetic molecular components of theGVR genetic circuit into the prokaryotic host.

According to an eighth aspect, a genetically engineered prokaryotic hostis described comprising one or more hybrid GVGC herein describedconfigured for expression in the genetically engineered prokaryotic hostand/or one or more GVR genetic circuits herein described configured foroperation in the genetically engineered prokaryotic host.

According to a ninth aspect, a composition is described. The compositioncomprises one or more genetic molecular components of a GVR geneticcircuit, vectors, or genetically engineered prokaryotic cells describedherein together with a suitable vehicle.

According to a tenth aspect, a method to provide a geneticallyengineered prokaryotic cell comprising one or more GVR genetic circuitsis described, the method comprising:

genetically engineering a prokaryotic cell by introducing into theprokaryotic cell one or more hybrid GVGC, hybrid GVGC genetic circuitsand/or GVGC genetic molecular components herein described.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems can beused in several embodiments for reporting biochemical events in aprokaryotic cell in vitro, or in vivo, and in particular can be used fornon-invasive reporting of biochemical events in prokaryotic cells usingimaging techniques such as MRI and ultrasound, two widely availabletechniques with high resolution and deep tissue penetration.

In several embodiments described herein, the hybrid GVGCs, and relatedGVR genetic circuits, vectors, genetically engineered prokaryotic cells,compositions, methods and systems can be used to report the location ofprokaryotic cells configured to express one or more GV types within animaging target site, and/or sense and report one or more biochemicalevents in prokaryotic cells configured to express one or more GV typeswithin an imaging target site.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to report one or morebiochemical events through magnetic resonance imaging with enhancedcontrast and molecular sensitivity down to sub-nanomolar concentration.

In particular, in several embodiments, gas vesicles (GVs) expressed ingenetically engineered prokaryotic cells comprising hybrid GVGCs and/orGVR genetic circuits described herein can be detected in the imagingtarget site using contrast-enhanced imaging techniques such as magneticresonance imaging (MRI) and ultrasound.

The hybrid GVGCs and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to allow multiplexedimaging using parametric MRI, and differential acoustic sensitivity andbackground-free MRI when combined with ultrasound.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to detect events such asmultiple gene expression, proteolysis and/or biochemical reactions byclustering-induced changes in MRI contrast also enable the design ofdynamic molecular sensors.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to report biochemicalevents through multiplexing, multimodal MRI and/or ultrasound detection.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to report biochemicalevents through non-toxic, robust MRI contrast via differential magneticsusceptibility at nanomolar concentrations.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to produce dynamic contrastin response to local molecular signals.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to provide ultrasoundimaging with enhanced harmonic responses, multiplexing, multimodaldetection and/or molecular targeting to help ultrasound fulfill itspotential as a high performance modality for molecular imaging.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems as wellas GvpC variants herein described can be used in several embodiments totrack movement in target sites such as prokaryotic cells within the bodyof an individual or other environments.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can in some embodiments be used to allow measures of fluidflows within blood and lymphatic circulation systems by detecting thespatial location of the ultrasound contrast produced the by the cells inan image and tracking the spatial changes of that contrast over time.

The hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in connection with various applications whereinreporting of biological events in a target site is desired. For example,the hybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used for visualization of biological events, such as agene expression, proteolysis, biochemical reactions as well asprokaryotic cell location on a target site (e.g. bacterial cells insidea host individual, such as mammalian hosts), facilitating for examplethe study of the mammalian microbiome and the development of diagnosticand therapeutic cellular agents, among other advantages identifiable bya skilled person, in medical applications, as well diagnosticsapplications. Additional exemplary applications include uses of thehybrid GVGCs, and related GVR genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed in several fields including basic biology research, appliedbiology, bio-engineering, bio-energy, medical research, medicaldiagnostics, therapeutics, and in additional fields identifiable by askilled person upon reading of the present disclosure.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description and theexamples, serve to explain the principles and implementations of thedisclosure.

FIG. 1 shows a rendition of GVs showing the related building blocks.GvpA ribs (1) (gray) forming the primary GV shell and the outer scaffoldprotein GvpC (2) (black dark rectangles (2).

FIG. 2 shows schematics and images showing exemplary genetic engineeringof GVGCs. Panel A shows a schematic of exemplary architecture of GVGCgene clusters. All clusters shown contain the B. megaterium genesGvpR-U, and vary in their composition of the structural genes GvpB (B.megaterium), GvpA and GvpC (A. floc-aquae). Three different constructsare shown, wherein each comprises a different set of GV structuralgenes, shown in the ‘zoomed-in’ view of the portion of the constructlabeled ‘A/B/C’. The construct on the left comprises GvpB (B.megaterium); the construct in the middle comprises GvpA (A. floc-aquae);the construct on the right comprises GvpA and GvpC (A. floc-aquae). Thestructural genes comprised in an exemplary GVGC referred to herein asAcoustic Reporter Gene 1 (ARG1) shown on the right, in which ARG1comprises A. floc-aquae structural genes GvpA and GvpC. Panels B-F areorganized in columns (left, middle, right) corresponding to each of theconstructs shown in Panel A. Panel B shows exemplary ultrasound imagesof agarose phantoms containing E. coli expressing each construct or GFP.The cell concentration is 10⁹ cells/ml. Images in bottom images of PanelB were acquired after acoustic collapse. Dotted outlines indicate thelocation of each specimen. The vertical bar on the right representslinear signal intensity of the images in Panel B (0-50). Panel C showsexemplary TEM images of representative E. coli cells expressing eachconstruct. Panel D shows exemplary TEM images of gas vesicles isolatedfrom E. coli expressing each construct. Panel E shows graphs reportingmass of GV proteins obtained from E. coli cultures expressing eachconstruct (N=3 per sample). Panel F shows exemplary images of E. coliexpressing each construct in liquid culture. Arrow points to themeniscus layer, where the ARG1 construct contains buoyant cells. Scalebars represent 2 mm in Panel B, 500 nm in Panel C and 250 nm in Panel D.Error bars represent±S.E.M.

FIG. 3 shows a schematic of exemplary sequence homology of GvpA/B. Theschematic shows amino acid sequence alignment of the primary gas vesiclestructural protein, GvpB from B. megaterium, showing the sequenceMSIQKSTNSSSLAEVIDRILDKGIVIDAFARVSVVGIEILTIEARVVIASVDTWLRYAEAVGLLRDDVEENGLPERSNSSEGQPRFSI (SEQ ID NO:1) and GvpA from A. floc-aquae,showing the sequenceMAVEKTNSSSSLAEVIDRILDKGIVIDAWVRVSLVGIELLAIEARIVIASVETYLKYAEAV GLTQSAAVPA(SEQ ID NO:2).

FIG. 4 shows exemplary results of imaging dilute bacterial populationsand dynamically regulated gene expression. Panel A shows exemplaryultrasound images of ARG1-expressing E. coli at various cellularconcentrations, before and after acoustic collapse. Panel B shows agraph reporting exemplary mean ultrasound contrast from E. coliexpressing ARG1 and GFP at various cell densities (N=4 per sample).Panel C shows exemplary ultrasound images of E. coli expressing ARG1after induction with various IPTG concentrations. Panel D shows a graphreporting exemplary normalized ultrasound contrast as a function of IPTGconcentration. Cell concentration was 5×10⁸ cells/ml. N=3 per sample.Panel E shows exemplary ultrasound images of ARG1-expressing E. coli atvarious times after induction with IPTG. Panel F shows a graph reportingexemplary mean ultrasound contrast at each time point (N=4 per sample).Cell concentration was 5×10⁸ cells/ml in Panels C-F. The vertical barson the right of each of Panels A, C and E represent linear signalintensity (0-50). Scale bars represent 2 mm. Error barsrepresent±S.E.M.; where not seen, they are smaller than the symbol.

FIG. 5 shows a schematic diagram and exemplary data of ultrasoundcontrast from buoyancy-enriched cells. Panel A shows a diagram ofcentrifugation-assisted enrichment of buoyant cells. Panel B showsexemplary ultrasound images of E. coli expressing ARG1 at variouscellular concentrations, with and without buoyancy enrichment. Panel Cshows a graph reporting exemplary mean ultrasound contrast from E. coliexpressing ARG1, with and without buoyant enrichment, and GFP at variouscell densities (N=4 per sample).

FIG. 6 shows exemplary data showing that GVGC expression and ultrasoundimaging does not affect cell viability. Panel A shows a graph reportingexemplary growth curves of E. coli containing the ARG1 or GFP expressionplasmid, with or without induction using 0.4 mM IPTG (N=3 per sample).Panel B shows exemplary representative TEM images of whole E. coli cellsexpressing ARG1 with and without exposure to acoustic collapse pulses,and E. coli cells expressing GFP. Panel C shows an exemplary dark fieldoptical image of agar plate containing colonies of E. coli expressingARG1 14 hours after seeding. Panel D shows an exemplary image of theplate shown in Panel C after the right half of the plate was insonatedwith high-pressure ultrasound. Panel E shows an exemplary image of theplate shown in Panels C and D 20 h after insonation. Panel F shows anexemplary image after the right half of the plate in Panel E wasinsonated with high-pressure ultrasound. Zoomed in images ofrepresentative colonies are shown below each plate image. Scale barsrepresent 500 nm. Error bars represent±S.E.M; where not seen, they aresmaller than the symbols.

FIG. 7 shows exemplary schematic diagrams and data of multiplexedimaging of genetically engineered reporter variants. Panel A shows adiagram of the exemplary GvpA and GvpC sequences included in the ARG1and ARG2 gene clusters. ARG2 was created by deleting 4 of the 5 repeatdomains in wild-type GvpC. Panel B shows exemplary ultrasound images ofa gel phantom containing E. coli expressing ARG2 or GFP (10⁹ cells/ml).Dotted outlines indicate the location of each specimen. Panel C showsexemplary transmission electron micrographs of isolated ARG2 gasvesicles. Panel D shows an exemplary image of ARG2 E. coli culture 22hours after induction showing the presence of buoyant cells and a graphreporting exemplary mass fraction of gas vesicles produced 22 hoursafter induction. (N=3). Panel E shows a graph reporting exemplarynormalized optical density (representing the intact fraction) of gasvesicles isolated from ARG1- or ARG2-expressing E. coli as a function ofapplied hydrostatic pressure (N=3 per sample). Panel F shows a graphreporting exemplary normalized ultrasound intensity as a function ofpeak positive pressure from 0.6 to 4.7 MPa for E. coli expressing ARG1or ARG2 (N=3 per sample). Panel G shows a graph reporting exemplaryacoustic collapse spectra derived by differentiating the data and curvesin Panel F with respect to applied pressure (N=3 per sample). Panel Hshows exemplary ultrasound images of gel phantoms containing ARG1 orARG2 before collapse, after collapse at 2.7 MPa and after collapse at4.5 MPa (10⁹ cells/mL). Panel I shows exemplary spectrally unmixed mapsof ARG2 and ARG1 obtained from the set of images in Panel H. Scale barsrepresent 2 mm in Panel B and Panels H-I and 250 nm in Panel C. Errorbars represent +/−S.E.M.

FIG. 8 shows graphs reporting exemplary ultrasound contrast from cellsexpressing ARG1 and ARG2. Panel A shows a graph reporting exemplaryultrasound contrast from the whole population of cells expressing ARG1,ARG2 or GFP (N=4 per sample). Panel B shows a graph reporting exemplaryultrasound contrast from the buoyancy-enriched population of cellsexpressing ARG1, ARG2 or GFP (N=3 per sample). Error bars represent±SEM.

FIG. 9 shows an exemplary illustration and exemplary data of highthroughput screening of acoustic phenotypes. Panel A shows an exemplaryillustration of acoustic colony screening. In the illustration, bacteriaplated on agar are scanned with an ultrasound transducer that collectsimages and applies various peak pressures for acoustic collapse. Animage of the 2D colony surface perpendicular to the transducer iscomputed from a series of contiguous images acquired in the transducer'simaging plane. Panel B shows exemplary colony ultrasound images of amixed population of ARG1, ARG2, and GFP expressing E. coli colonies.Images were acquired before collapse and after collapse at 4.0 and 6.0MPa peak acoustic pressures. Panel C shows an exemplary image ofpredicted genotypes of each colony based on the acoustic phenotype seenin the images in Panel B. Panel D shows an exemplary ultrasoundintensity histogram of 22 randomly picked colonies. Colonies with lowcontrast were predicted to be GFP and those with high contrast to beARG1 or ARG2. Panel E shows a graph reporting normalized change inultrasound intensity for each of the randomly picked colonies afterinsonation at increasing pressures. At 4 MPa, colonies with signal abovethe indicated threshold were predicted to be ARG1 and below to be ARG2.Panel F shows an exemplary graph reporting confirmation by sequencing ofpredicted genotypes indicated in Panels D and E. Scale bar represents 10mm.

FIG. 10 shows exemplary diagrams and data of ultrasound imaging ofbacteria in the gastrointestinal tract. Panel A shows an exemplarydiagram of GI imaging experiment. E. coli expressing ARG2 wereintroduced into the colon of mice and imaged with ultrasound. Panel Bshows exemplary transverse ultrasound images of mice whose coloncontains E. coli expressing either ARG2 or GFP at a final concentrationof 10⁹ cells/ml. A difference heat map of ultrasound contrast within thecolon ROI (circled region of interest indicated with arrow) before andafter acoustic collapse is overlaid on an anatomical image. Panel Cshows a graph reporting exemplary average signal intensity of ultrasoundcontrast within the colon ROI in mice with E. coli expressing eitherARG2 or GFP. N=5 mice per sample. Scale bar represents 2.5 mm in PanelB. Error bars represent ±SEM.

FIG. 11 shows exemplary anatomical ultrasound images of bacteriaexpressing GVGC in the gastrointestinal tract. Shown are raw imagesunderlying the difference map shown in FIG. 10. The circle outlineidentifies the colon region of interest for difference processing.

FIG. 12 shows exemplary ultrasound imaging of S. typhimurium in tumorxenografts. Panel A shows exemplary ultrasound images of a gel phantomcontaining S. typhimurium expressing ARG1 or the LuxABCDE operon. Cellconcentration is 10⁹ cells/ml. Panel B shows exemplary representativeTEM images of whole S. typhimurium cells expressing ARG1 with andwithout exposure to acoustic collapse pulses. Panel C shows exemplaryrepresentative ultrasound images of mouse OVCAR8 tumors injected with 50μL of 3.2×10⁹ cells/ml ARG1-expressing S. typhimurium, before and afteracoustic collapse. Panel D shows a graph reporting exemplary meancollapse-sensitive ultrasound contrast in N=5 tumors injected withARG1-expressing or LuxABCDE-expressing cells. Scale bars 500 nm in PanelB and 2.5 mm in Panel C. Error bars represent ±SEM.

FIG. 13 shows exemplary data of colony ultrasound of E. coli expressingARG1 or ARG2. Panel A shows exemplary ultrasound images of platescontaining either ARG1 or ARG2 expressing E. coli colonies. Images wereacquired before collapse and after collapse at the indicated peakacoustic pressures. Panel B shows a graph reporting exemplary normalizedaverage colony change in ultrasound intensity after insonation atincreasing pressures (N=15 per sample). All error bars represent +/−SEM.The crosshairs indicate determined thresholds for distinguishing ARG1 vsARG2 based on acoustic phenotype. Scale bar 10 mm. Error bars represent±S.E.M.

FIG. 14 shows the plasmid sequence of the exemplary ARG1 construct.

FIG. 15 shows the plasmid sequence of the exemplary ARG2 construct.

FIG. 16 shows exemplary acoustomagnetic reporter gene imaging in livingcells.

Panel A shows a schematic diagram of exemplary inducible expression ofGVGCs in E. coli leading to the intracellular formation of GVs and thegeneration of susceptibility-based MRI contrast. Panel B shows anexemplary representative acoustomagnetic QSM image of agarose phantomcontaining E. coli expressing GVGCs or a green fluorescent protein (GFP)under the control of an IPTG-inducible promoter, in the presence orabsence of the inducer, compared to a well containing buffer. Panel Cshows a graph reporting exemplary mean differential susceptibilityvalues relative to buffer. N=6 biological replicates. Error barsrepresent SEM. All bacterial cells were at culture OD₆₀₀=8.0.

FIG. 17 shows a graph reporting exemplary hydrostatic collapsemeasurement of E. coli cells. E. coli cells at optical density at 600 nm(OD600)˜1.0 were loaded into a sealed cuvette with path length 1.00 cm.Hydrostatic pressure was ramped stepwise from 0 to 1.2 MPa and OD600 wasrecorded in each step. Cells expressing exemplary A2C GVs showed asigmoidal drop in OD600, characteristic of the collapse of intracellularGV. Cells that do not contain GV, such as the control cells expressingthe green fluorescent protein (GFP) mNeonGreen5 did not show a drop inOD600. The ratio of post- to pre-collapse optical density was between0.806 and 0.853 (Min and Max, N=6), and this ratio was used to adjustthe OD600 GV-expressing cells to be representative of cell quantity.

FIG. 18 shows an exemplary Clustal omega alignment of amino acidsequences of selected exemplary gvpA and gvpB proteins. The gvpA andgvpB proteins shown are from the following species: Sa_A2, Serratia sp.ATCC 39006 gvpA2; Sa_A3, Serratia sp. ATCC 39006 gvpA3; Sc_A2,Streptomyces coelicolor gvpA2; Sc_A1, Streptomyces coelicolor gvpA1;Fc_A, Frankia sp. gvpA; Bm_B1, B. megaterium gvpB1; Mb_A, Methanosarcinabarkeri gvpA; Hv_A, Halorubrum vacuolatum gvpA; Hm_A, Haloferaxmediterranei gvpA; Hs_A1, Halobacterium sp. NRC-1 gvpA1; Hs_A2,Halobacterium sp. NRC-1 gvpA2; Bm_A, B. megaterium gvpA; Bm_B2, B.megaterium gvpB2; Af_A, A. floc-aquae gvpA; Ma_A, Microcystis aeruginosaNIES-843 gvpA; Sa_A1, Serratia sp. ATCC 39006 gvpA1. The bottom row ofFIG. 18 indicated as “Consensus” shows an exemplary consensus sequencederived from alignment of the gvpA and gvpB amino acid sequences shown.

FIG. 19 shows an exemplary configuration of a construct designed toallow expression of two different GV types in one prokaryotic cell. Theexemplary construct in FIG. 19 is designed to provide alternatingexpression of two GV types, the first GV type encoded by Cluster 1, andthe second GV type encoded by Cluster 2, shown as block-shaped arrowsfacing in opposite orientations of a DNA strand (shown as a straightline), with a promoter between the two clusters. The promoter is flankedby recombination sites (e.g. flippase recognition target, FRT sites)shown as circles. For example, initially, the promoter can be orientedin a direction operatively linked to Cluster 1, initiating expression ofgyp genes for the formation of GV type 1. In presence of a cognaterecombinase (e.g. flippase, Flp), expressed from another geneticconstruct in the prokaryotic cell, the orientation of the promoter isreversed upon recombination at the FRT sites, and thereafter is orientedin the opposite direction, operatively linked to Cluster 2, initiatingexpression of gyp genes for the formation of GV type 2.

FIG. 20 shows diagrams illustrating the organization of exemplary gasvesicle gene clusters. Gas vesicle gene clusters from the indicatedorganisms are shown, with genes shown as block-shaped arrows, and genesof predicted similar function indicated in the same shade of grey. Thedirection of the transcription of genes within a gene cluster isindicated by the direction of the block-shaped arrows, and genes groupedtogether having block arrows pointed in the same direction are typicallyorganized in the same operon. The scale bar indicates 1 kb. [1]

FIG. 21 shows diagrams illustrating organization of exemplary Gvp geneclusters, wherein each letter indicates a Gvp gene, and an arrow beneatha group of letters indicates an operon, with the direction of the arrowindicating the direction of transcription. [2]

FIG. 22 shows exemplary phylogenetic relationships of the gvpA proteinsequences from the indicated prokaryotic species. [1]

FIG. 23 shows exemplary phylogenetic relationships of the gvpF and gvpLprotein sequences from the indicated prokaryotic species. [1]

FIG. 24 shows exemplary phylogenetic relationships of the gvpN proteinsequences from the indicated prokaryotic species. [1]

DETAILED DESCRIPTION

Provided herein are genetically engineered gas vesicle gene clusters(GVGC), and related gas vesicles (GVs), genetic circuits, vectors,genetically engineered prokaryotic cells, compositions, methods andsystems.

The wordings “gas vesicles”, GV″, “gas vesicles protein structure”, or“GVPS”, refer to a gas-filled protein structure natively intracellularlyexpressed by certain bacteria or archea as a mechanism to regulatecellular buoyancy in aqueous environments [3]. In particular, gasvesicles are protein structures natively expressed almost exclusively inmicroorganisms from aquatic habitats, to provide buoyancy by loweringthe density of the cells [3]. GVs have been found in over 150 species ofprokaryotes, comprising cyanobacteria and bacteria other thancyanobacteria [4, 5], from at least 5 of the 11 phyla of bacteria and 2of the phyla of archaea described by Woese (1987) [6]. Exemplarymicroorganisms expressing or carrying gas vesicle protein structuresand/or related genes include cyanobacteria such as Microcystisaeruginosa, Aphanizomenon flos aquae Oscillatoria agardhii, Anabaena,Microchaete diplosiphon and Nostoc; phototropic bacteria such asAmoebobacter, T hiodiclyon, Pelodiclyon, and Ancalochloris; nonphototropic bacteria such as Microcyclus aquaticus; Gram-positivebacteria such as Bacillus megaterium Gram-negative bacteria such asSerratia; and archaea such as Haloferax mediterranei, Methanosarcinabarkeri, and Halobacteria salinarium, as well as additionalmicroorganisms identifiable by a skilled person.

In particular, a GV in the sense of the disclosure is an intracellularlyexpressed structure forming a hollow structure wherein a gas is enclosedby a protein shell, which is a shell substantially made of protein (atleast 95% protein). In gas vesicles in the sense of the disclosure, theprotein shell is formed by a plurality of proteins herein also indicatedas Gvp proteins or Gvps, which form in the cytoplasm a gas permeable andliquid impermeable protein shell configuration encircling gas.Accordingly, a protein shell of a GV is permeable to gas but not tosurrounding liquid such as water. In particular, GV protein shellsexclude water but permit gas to freely diffuse in and out from thesurrounding media [7] making them physically stable despite their usualnanometer size, unlike microbubbles, which trap pre-loaded gas in anunstable configuration.

Gvp proteins natively expressed by prokaryotes such as bacteria orarchea and forming the protein shell of a GV are also indicated as GasVesicle Structural (GVS) proteins.

The term Gas Vesicle structural (GVS) proteins as herein indicatesproteins forming part of a gas-filled protein structure intracellularlyexpressed by certain bacteria or archea and can be used as a mechanismto regulate cellular buoyancy in aqueous environments [7]. In particularGVS shell comprises a GVS identified as gvpA or gvpB (herein alsoreferred to as Gvp A/B) and optionally also a GVS identified as GvpC.

In particular, a gyp A/B is a protein of the GV shell that has a higherthan 70% identity to the following consensus sequence:SSSLAEVLDRILDKGXVIDAWARVSLVGIEILTIEARVVIASVDTYLR (SEQ ID NO: 3) whereinX can be any amino acid. In particular in a gvpA/B of prokaryotes, theconsensus sequence of SEQ ID NO: 3 typically forms a conserved secondarystructure having an alpha-beta-beta-alpha structural motif formed byportions of the consensus sequence comprising the amino acids LDRILD(SEQ ID NO:4) having an alpha helical structure, RILDKGXVIDAWARVS (SEQID NO:5) wherein X can be any amino acid, having a beta strand, betastrand structure, and DTYLR (SEQ ID NO:6) having an alpha helicalstructure, as will be understood by a skilled person.

Thus, a gvpA/B protein in a prokaryote of interest can be identified forexample by isolating GVs from a prokaryote of interest, isolating theprotein from the protein shell of the GV and obtaining the amino acidsequence of the isolated protein. In addition or in the alternative tothe isolating the GVs and isolating the protein, the method can includeobtaining amino acidic sequences of the shell proteins of the GV of theprokaryote of interest from available database. The method furthercomprises performing a sequence alignment of the obtained amino acidicsequences against the gvpA/B protein consensus sequence of SEQ ID NO:3.

In particular the isolating GVs from a prokaryote of interest can beperformed following methods to isolate gas vesicles as described in U.S.application Ser. No. 15/613,104, filed on Jun. 2, 2017. The isolatingthe protein for the protein shell of the GV and obtaining the relatedamino acidic sequence can be performed with tandem liquid chromatographymass-spectrometry alone or in combination with obtaining amino acidsequences of the isolated protein with wet lab techniques or fromavailable databases comprising the sequences of the prokaryote ofinterest as well as additional techniques and approaches identifiable bya skilled person. Obtaining amino acid sequences of GV shell proteins ofthe prokaryote of interest can be performed by screening availabledatabases of gene and protein sequences identifiable by a skilledperson. Performing a sequence alignment of the sequences of the isolatedGV proteins or proteins encoded in the genome of a prokaryote ofinterest can be performed (using Protein BLAST or other alignmentalgorithms known in the art) against the gvpA/B protein consensussequence of SEQ ID NO:3. In particular, a sequence alignment can beperformed using gvpA/B protein sequences from the closest phylogeneticrelative to the prokaryote of interest. Reference is made to Example 13and FIG. 22 showing exemplary phylogenetic relationships between gvpA/Bproteins of exemplary prokaryotic species.

A GvpC protein is a hydrophilic protein of a GV shell, which includesrepetitions of one repeat region flanked by an N-terminal region and a Cterminal region. The term “repeat region” or “repeat” as used hereinwith reference to a protein refers to the minimum sequence that ispresent within the protein in multiple repetitions along the proteinsequence without any gaps. Accordingly, in a GvpC multiple repetitionsof a same repeat is flanked by an N-terminal region and a C-terminalregion. In a same GvpC, repetitions of a same repeat in the GvpC proteincan have different lengths and different sequence identity one withrespect to another.

Repeat regions within any given GvpC sequence ‘X’ from organism ‘Y’ canbe identified by comparing the related sequence with the sequence of aknown GvpC (herein e.g. reference GvpC sequence “Z”). In particular, thecomparing can be performed by aligning sequence ‘X’ to the referenceGvpC sequence ‘Z’ using a sequence alignment tools such as BLASTP orother sequence alignment tools identifiable by a skilled person at thedate of filing of the application upon reading of the presentdisclosure. In particular, a reference sequence ‘Z’ is chosen from ahost that is the closest phylogenetic relative of ‘Y’, from a list ofAnabaena flos-aquae, Halobacterium salinarum, Haloferax mediditerranei,Microchaetae diplosiphon and Nostoc sp. The sequence alignment of ‘X’and ‘Z’ (e.g. a BLASTP) is performed by performing a first alignment ofsequence X and sequence Z to identify a beginning and an end of a repeatin ‘X as well as a number of repetition of the identified repeat, inaccordance with the known repeats in ‘Z’. The first alignment results inat least one first aligned portion of X with respect to referencesequence Z. The aligning can also comprises performing a secondalignment between the at least one first aligned portion of X identifiedfollowing the first alignment and additional portions of X to identifyat least one repeat ‘R1’ in X. Other repeats in ‘X’ (i.e. R2, R3, R4 . .. ) can subsequently be identified with respect to R1.

In performing alignment steps sequence are identified as repeat when thesequence shows at least 3 or more of the following characteristics:

-   1) There are no gaps or spacer amino acids between any two adjacent    repetition of a repeat (see e.g. FIG. 16 and FIG. 26 of U.S.    application Ser. No. 15/613,104 filed on Jun. 2, 2017)-   2) Each repetition of a repeat has a sequence length between 18-45    amino acids, e.g. 33 amino acids seen for 100% of the repeats in    Anabaena floc-aquae, Microchaetae diplosiphon and Nostoc sp. (e.g.    FIG. 26 of U.S. application Ser. No. 15/613,104 filed on Jun. 2,    2017)-   3) Upon alignment of all the repeats within a given GvpC sequence,    there exists for every position in more than 50% of the total number    of repeats, greater than 50% sequence similarity of the amino acid    residues in each repeat (e.g. FIG. 26 of U.S. application Ser. No.    15/613,104 filed on Jun. 2, 2017)-   4) Sub-sequences of at least 3 or more amino acids at the beginning    or end of the that are conserved across 50% or more of the repeats    in a given GvpC sequence, also referred to as “consensus sequences”.    Exemplary embodiments of such consensus sequences are QAQELLAF (SEQ    ID NO:7) at the end of repeats in Anabaena floc-aquae, LHQF (SEQ ID    NO:8) at the end of repeats in Microchaete diplosiphon, LSQF (SEQ ID    NO:9) at the end of repeats in Microcystis aeruginosa and DAF (SEQ    ID NO:10) at the beginning of repeats in Halobacterium salinarum.    (e.g. FIGS. 16 and 26 of U.S. application Ser. No. 15/613,104 filed    on Jun. 2, 2017).-   5) The consensus sequence of all the repeats within a given GvpC    sequence show greater than 60% identity to the consensus sequence of    all the repeats within another GvpC from a different microbial host    of the same phylogenetic order (e.g. FIG. 26, panels g-h of U.S.    application Ser. No. 15/613,104 filed on Jun. 2, 2017).

In some exemplary embodiments, the repeat has at least 90% sequenceidentity with another repeat within the same GvpC sequence.

In a GvpC the N-terminal region comprises the amino acid residuesupstream (towards the N-terminus) of the first repeated sequence of theGvpC's repeat, while the C-terminal region comprises the amino acidresidues downstream (towards the C-terminus) of the last repeatedsequence of the GvpC's repeat.

GvpC protein is typically rich in glutamine, alanine and glutamic acidresidues, which account for >40% of the residues. In the exemplaryAnabaena flos-aqaue, GvpC comprises five highly conserved 33-amino acidrepeats with predicted alpha-helical structure, and is believed to bindacross GvpA ribs to provide structural reinforcement [3], which alignswith experimental data. In biochemical studies, removal of GvpC andtruncations to its sequence were shown to result in a reduced thresholdfor Ana GV collapse under hydrostatic pressure. In addition, previousstudies in other species have demonstrated that GvpC can toleratefusions of bacterial and viral polypeptides.

GvpC sequences in different bacteria or archaea producing GVs typicallyhave a greater than 15% sequence identity and are produced by genesfound in the gas vesicle gene cluster.

Following purification of GVs from a bacteria or archea naturallyexpressing the GV, GVS proteins make up over 90% of the bulk GV mass.

In embodiments herein described GVS proteins natively expressed bybacteria or archea assemble in the native bacterial or archeal cell inpresence of additional proteins also natively expressed in the nativebacterial or archeal cells herein also indicated as Gas Vesicle Assembly(GVA) proteins which putative minor components and chaperones [8-10] aswould be understood by a person skilled in the art.

The term Gas Vesicle Assembly (GVA) proteins as used herein indicatesproteins enabling assembly of a GV in a prokaryotic cell. GVA proteinscomprise proteins with various putative functions such as nucleatorsand/or chaperons as well as proteins with an unknown specific functionrelated to the assembly of the GV.

Accordingly, in bacterial and archeal cells natively expressing GVS, inpresence of natively expressed GVAs, GvpA/B assemble through repeatedunits to make up the bulk of GVs, while GvpC provides a scaffold proteinwith repeat units that assemble on the outer shell of GVs.

Reference is made to the illustration of FIG. 1 showing a schematicrepresentation of the structure of an exemplary GV. In the illustrationof FIG. 1 GvpA and GvpC are indicated as the two major structuralconstituents of GVs, with GvpA ribs (1) (gray) forming the primary GVshell and the outer scaffold protein GvpC (2) (black) conferringstructural integrity. In particular, in the illustration of FIG. 1, thelight gray elements represent the proteinaceous gas vesicle shell,comprising multiple copies of GvpA and other minor structuralconstituents. In the illustration of FIG. 1, the dark rectangles (2)bound to the surface of the gas vesicle shell represent GvpC, a proteinthat affects mechanical and acoustic properties of the gas vesicle.

GV structures are typically nanostructures with widths and lengths ofnanometer dimensions (in particular with widths of 45-250 nm and lengthsof 100-800 nm) but can have lengths up to 2 μm as will be understood bya skilled person. In certain embodiments, the gas vesicles proteinstructure have average dimensions of 1000 nm or less, such as 900 nm orless, including 800 nm or less, or 700 nm or less, or 600 nm or less, or500 nm or less, or 400 nm or less, or 300 nm or less, or 250 nm or less,or 200 nm or less, or 150 nm or less, or 100 nm or less, or 75 nm orless, or 50 nm or less, or 25 nm or less, or 10 nm or less. For example,the average diameter of the gas vesicles may range from 10 nm to 1000nm, such as 25 nm to 500 nm, including 50 nm to 250 nm, or 100 nm to 250nm. By “average” is meant the arithmetic mean.

GVs in the sense of the disclosure have different shapes depending ontheir genetic origins [7]. For example, GVs in the sense of thedisclosure can be substantially spherical, ellipsoid, cylindrical, orhave other shapes such as football shape or cylindrical with cone shapedend portions depending on the type of bacteria providing the gasvesicles.

Representative examples of endogenously expressed GVs native tobacterial or archaeal species are the gas vesicle protein structureproduced by the Cyanobacterium Anabaena flos-aquae (Ana GVs) [3], andthe Halobacterium Halobacterium salinarum (Halo GVs) [10]. Inparticular, Ana GVs are cone-tipped cylindrical structures with adiameter of approximately 140 nm and length of up to 2 μm and inparticular 200-800 nm or longer, encoded by a cluster of nine differentgenes, including the two primary structural proteins, GvpA and GvpC, andseveral putative minor components and putative chaperones[11] as wouldbe understood by a person skilled in the art. Halo GVs are typicallyspindle-like structures with a maximal diameter of approximately 250 nmand length of 250-600 nm, encoded by a cluster of fourteen differentgenes, including the two primary structural proteins, GvpA and GvpC, andseveral putative minor components and putative chaperones [11] as wouldbe understood by a person skilled in the art.

In bacteria or archaea expressing GVs, the Gvp structural proteinsforming a GV's protein shell and Gvp assembly proteins allowing assemblyof the GVS proteins into a shell, are encoded by a gas vesicle genecluster of 8 to 14 different genes depending on the host bacteria orarchaea, as will be understood by a skilled person.

The term “Gas Vesicle Genes Cluster” or “GVGC” as described hereinindicates a gene cluster encoding a set of Gvp proteins capable ofproviding a GV upon expression within a bacterial cell.

The term “gene cluster” as used herein means a group of two or moregenes found within an organism's DNA that encode two or morepolypeptides or proteins, which collectively share a generalizedfunction or are genetically regulated together to produce a cellularstructure and are often located within a few thousand base pairs of eachother. The size of gene clusters can vary significantly, from a fewgenes to several hundred genes [12]. Portions of the DNA sequence ofeach gene within a gene cluster are sometimes found to be similar oridentical; however, the resulting protein of each gene is distinctivefrom the resulting protein of another gene within the cluster. Genesfound in a gene cluster can be observed near one another on the samechromosome or native plasmid DNA, or on different, but homologouschromosomes. An example of a gene cluster is the Hox gene, which is madeup of eight genes and is part of the Homeobox gene family. In the senseof the disclosure, gene clusters as described herein also comprise gasvesicle gene clusters, wherein the expressed proteins thereof togetherare able to form gas vesicles.

In embodiments herein described identification of a gene clusterencoding Gvp proteins naturally expressed in bacteria or archea asdescribed herein can be performed for example by isolating the GVs fromthe bacteria or archea, isolating the protein for the protein shell ofthe GV and deriving the related amino acidic sequence with methods andtechniques identifiable by a skilled person. The sequence of the genesencoding for the Gvp proteins can then be identified by methods andtechniques identifiable by a skilled person. For example, gas vesiclegene clusters can also be identified by persons skilled in the art byperforming gene sequencing or partial- or whole-genome sequencing oforganisms using wet lab and in silico molecular biology techniques knownto those skilled in the art. As understood by those skilled in the art,gas vesicle gene clusters can be located on the chromosomal DNA ornative plasmid DNA of microorganisms. After performing DNA or cDNAisolation from a microorganism, the polynucleotide sequences orfragments thereof or PCR-amplified fragments thereof can be sequencedusing DNA sequencing methods such as Sanger sequencing, DNASeq, RNASeq,whole genome sequencing, and other methods known in the art usingcommercially available DNA sequencing reagents and equipment, and thenthe DNA sequences analyzed using computer programs for DNA sequenceanalysis known to skilled persons.

In some embodiments, identification of a gene cluster encoding for Gvpproteins [8-10] can also be performed by screening DNA sequencedatabases such as GenBank, EMBL, DNA Data Bank of Japan, and others. Gasvesicle gene cluster gene sequences in databases such as those above canbe searched using tools such as NCBI Nucleotide BLAST and the like, forgas vesicle gene sequences and homologs thereof, using gene sequencequery methods known to those skilled in the art. For example, genes ofthe gene cluster for the exemplary haloarchael GVs (which have thelargest number of different gyp genes) and their predicted function andfeatures are illustrated in Example 26 of related U.S. application Ser.No. 15/613,104, filed on Jun. 2, 2017 which is incorporated herein byreference in its entirety.

A GV gene cluster encoding for Gvp proteins typically comprises GasVesicle Assembly (GVA) genes and Gas Vesicle Structural (GVS) genes.

The Gas Vesicle Assembly genes are genes encoding for GVA proteins. In aprokaryotic cell GVA genes are all the genes within one or more operonscomprising at least one of a GvpN and a GvpF excluding any GvpA/B andGvpC gene possibly present within said one or more operons.

In particular, gvpN gene in the sense of the disclosure is gene encodingfor sequence

(SEQ ID NO: 11) MTVLTDKRKKGSGAFIQDDETKEVLSRALSYLKSGYSIHFTGPAGGGKTSLARALAKKRKRPVMLMHGNHELNNKDLIGDFTGYTSKKVIDQYVRSVYKKDEQVSENWQDGRLLEAVKNGYTLIYDEFTRSKPATNNIFLSILEEGVLPLYGVKMTDPFVRVHPDFRVIFTSNPAEYAGVYDTQDALLDRLITMFIDYKDIDRETAILTEKTDVEEDEARTIVTLVANVRNRSGDENSSGLSLRASLMIATLATQQDIPIDGSDEDFQTLCIDILHHPLTKCLDEENAKSKAEK IILEECKNIDTEEKor a sequence of any length having at least 30% sequence identity withrespect to SEQ ID NO:11, preferably at least 50%, and more preferably60% or higher,and gvpF gene in the sense of the disclosure is gene encoding forsequence

(SEQ ID NO: 12) MSETNETGIYIFSAIQTDKDEEFGAVEVEGTKAETFLIRYKDAAMVAAEVPMKIYHPNRQNLLMHQNAVAAIMDKNDTVIPISFGNVFKSKEDVKVLLENLYPQFEKLFPAIKGKIEVGLKVIGKKEWLEKKVNENPELEKVSASVKGKSEAAGYYERIQLGGMAQKMFTSLQKEVKTDVFSPLEEAAEAAKANEPTGETMLLNASFLINREDEAKFDEKVNEAHENWKDKADFHYSGPWPAYNF VNIRLKVEEK,or a sequence of any length having at least 20% sequence identity withrespect to SEQ ID NO:12, preferably at least 50%, more preferably 60%,and at least 70% or higher.

The term “operon” as described herein indicates a group of genesarranged in tandem in a prokaryotic genome as will be understood by askilled person. Operons typically encode proteins participating in acommon pathway are organized together as understood by those skilled inthe art. Typically, genes of an operon are transcribed together into asingle mRNA molecule referred to as polycistronic mRNA. PolycistronicmRNA comprises several open reading frames (ORFs), each of which istranslated into a polypeptide. These polypeptides usually have a relatedfunction and their coding sequence is grouped and regulated together ina regulatory region, containing a promoter and an operator. Typically,repressor proteins bound to the operator sequence can physicallyobstruct the RNA polymerase enzyme from binding the promoter, preventingtranscription. An example of a prokaryotic operon is the lac operon,which natively regulates transport and metabolism of lactose in E. coliand many other enteric bacteria.

In an operon, each ORF typically has its own ribosome binding site (RBS)so that ribosomes simultaneously translate ORFs on the same mRNA. Someoperons also exhibit translational coupling, where the translation ratesof multiple ORFs within an operon are linked. This can occur when theribosome remains attached at the end of an ORF and translocates along tothe next ORF without the need for a new RBS. Translational coupling isalso observed when translation of an ORF affects the accessibility ofthe next RBS through changes in RNA secondary structure.

In some embodiments, a GV cluster comprises one of gvpN or gvpF. Inseveral embodiments GV clusters include both gvpN and gvpF as will beunderstood by a skilled person. In this connection, reference is made toExample 12 and FIGS. 20 and 21 showing exemplary gas vesicle geneclusters operons [1, 2] comprising GVS and GVA genes and relatedexemplary configuration. In particular, as shown in Example 12,typically a native GV gene cluster has GVA genes comprising both gvpNand gvpF genes, even if native GV gene clusters are known having a gvpNgene or a gvpF gene, as understood by skilled persons.

Accordingly, for a certain prokaryote, GVA genes in the sense of thedisclosure indicate all the genes that are comprised in the one or moreoperons having at least one of a GvpN and/or a GvpF herein described andexcluding any Gas Vesicle Structural (GVS) genes of the prokaryotespossibly comprised within the one or more operons. Thus, GVA genescomprised in a gas vesicle gene cluster in a prokaryote can beidentified for example by obtaining genome sequence of the prokaryote ofinterest and performing a sequence alignment of the protein sequencesencoded in the genome of the prokaryote of interest against a gvpNprotein sequence and/or a gvpF protein sequence.

In particular, obtaining the genome sequence of the prokaryote ofinterest, can be performed either using wet lab techniques identifiableby a skilled person upon reading of the present disclosure, or obtainedfrom databases of gene and protein sequences also identifiable by askilled person upon reading of the present disclosure. Performing asequence alignment of the protein sequences encoded in the genome of theprokaryote of interest can per performed using Protein BLAST or otheralignment algorithms identifiable by a skilled person. Exemplary gvpNprotein sequence and/or a gvpF protein sequence, that can be used inperforming the alignment are sequences SEQ ID NO:11 and/or SEQ ID NO:12.In particular, a sequence alignment can be performed using gvpN and/orgvpF protein sequences from the closest phylogenetic relative to theprokaryote of interest. Reference is made to Example 13 and FIG. 23 andFIG. 24 showing exemplary phylogenetic relationships between gvpF andgvpN proteins of exemplary prokaryotic species. Accordingly, one or moreoperons that comprise the gvpN and/or gvpF genes can be identified, andany other gyps within the one or more operons can also be identified,wherein the other gyps are comprised in ORFs within the one or moreoperons, excluding any ORFs encoding gvpA/B or gvpC genes comprised inthe one or more operons of the GV gene cluster.

The Gas Vesicle Structural (GVS) genes are genes encoding for GVSproteins of a prokaryote as will be understood by a skilled person. In aprokaryotic cell GVS genes are genes within one or more operons that canbe identified as described herein with reference to the consensus aminoacid sequence of the encoded gvpA/B protein and gvpC protein sequences.As understood by skilled persons, in different species endogenous GVSgenes are natively located at varying positions within the one or moreoperons of a GV gene cluster (e.g. see FIGS. 20 and 21).

Some prokaryotic cells, such as Bacillus Megaterium natively include GVAgenes and GVS genes without however natively expressing said genes toprovide natively expressed GV.

In embodiments herein described, a GVGC is designed to include GVA genesand GVS genes in a configuration allowing co-expression of these genesin a host prokaryotic cell.

The term “prokaryotic” is used herein interchangeably with the terms“cell” or “host” refers to a microbial species which contains no nucleusor other organelles in the cell, which includes but is not limited toBacteria and Archaea.

The term “bacteria” as used herein refers to several prokaryoticmicrobial species which include but are not limited to Gram-positivebacteria, Proteobacteria, Cyanobacteria, Spirochetes and relatedspecies, Planctomyces, Bacteroides, Flavobacteria, Chlamydia, Greensulfur bacteria, Green non-sulfur bacteria including anaerobicphototrophs, Radioresistant micrococci and related species, Thermotogaand Thermosipho thermophiles. More specifically, the wording “Grampositive bacteria” refers to cocci, nonsporulating rods and sporulatingrods, such as, for example, Actinomyces, Bacillus, Clostridium,Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium,Myxococcus, Nocardia, Staphylococcus, Streptococcus and Streptomyces.The term “Proteobacteria” refers to purple photosynthetic andnon-photosynthetic gram-negative bacteria, including cocci, nonentericrods and enteric rods, such as, for example, Neisseria, Spirillum,Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella,Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Pseudomonas,Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter,Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponemaand Fusobacterium. Cyanobacteria, e.g., oxygenic phototrophs;

The term “Archaea” as used herein refers to prokaryotic microbialspecies of the division Mendosicutes, such as Crenarchaeota andEuryarchaeota, and include but is not limited to methanogens(prokaryotes that produce methane); extreme halophiles (prokaryotes thatlive at very high concentrations of salt (NaCl); and extreme (hyper)thermophiles (prokaryotes that live at very high temperatures).

In some embodiments the prokaryotic host is a bacteria and in particulara Gram Negative Bacteria. As understood by those skilled in the art,Gram-negative bacteria are a group of bacteria that do not retain thecrystal violet stain used in the Gram staining method of bacterialdifferentiation. They are characterized by their cell envelopes, whichare composed of a thin peptidoglycan cell wall sandwiched between aninner cytoplasmic cell membrane and a bacterial outer membrane.

Exemplary Gram-negative bacteria that can be genetically engineered withGVGC genetic circuits described herein configured to allow heterologousexpression of GVs comprise E. coli, Nissle 1997, Salmonella, and othersidentifiable by those skilled in the art.

In particular, in embodiments herein described, GVGC herein describedcomprise GVA genes and GVS genes in a configuration allowingco-expression of the gas vesicle structural genes and the gas vesicleassembly factor genes.

In particular in some embodiments the GVA genes and GVS genes are in oneor more polynucleotides at a variable distance one with respect toanother with polynucleotides sequences in between GVA and GVS which canvary and are configured to allow co-expression of all the GVA genes andall the GVS genes in one or multiple transcripts upon operativeconnection with one or more regulatory sequences.

The term “regulatory sequence” or “regulatory regions” as describedherein indicate a segment of a nucleic acid molecule which is capable ofincreasing or decreasing transcription or translation of a gene withinan organism either in vitro or in vivo. In particular coding regions ofa GVA genes and GVS genes herein described comprise one or more proteincoding regions which when transcribed and translated produce apolypeptide. Regulatory regions of a gene herein described comprisepromoters, transcription factor binding sites, operators, activatorbinding sites, repressor binding sites, enhancers, protein-proteinbinding domains, RNA binding domains, DNA binding domains, silencers,insulators and additional regulatory regions that can alter geneexpression in response to developmental and/or external stimuli as willbe recognized by a person skilled in the art.

The term “operative connection” as used herein indicate an arrangementof elements in a combination enabling production of an appropriateeffect. With respect to genes and regulatory sequences an operativeconnection indicates a configuration of the genes with respect to theregulatory sequence allowing the regulatory sequences to directly orindirectly increase or decrease transcription or translation of thegenes.

In particular, in some embodiments, regulatory sequences directlyincreasing transcription of the operatively linked gene or gene cluster,comprise promoters typically located on a same strand and upstream on aDNA sequence (towards the 5′ region of the sense strand), adjacent tothe transcription start site of the genes whose transcription theyinitiate. In prokaryotic organisms, promoters typically comprise twoshort DNA sequences located at the −10 (10 bp upstream) and −35positions from the transcription start site (TSS). Their equivalent tothe eukaryotic TATA (SEQ ID NO:13) box, the Pribnow box (TATAAT (SEQ IDNO:14)) is located at the −10 position and is essential fortranscription initiation. The −35 position, also referred to as the −35element, typically consists of the sequence TTGACA (SEQ ID NO:15) andthis element controls the rate of transcription. Prokaryotic cellscontain sigma factors which assist the RNA polymerase in binding to thepromoter region. Each sigma factor recognizes different core promotersequences identifiable by those skilled in the art. Thus, in severalembodiments described herein, promoters comprising binding sites forsigma factors identifiable by those skilled in the art can be used toregulate expression of the GVGC herein described in prokaryotic cells.

In some embodiments regulatory sequences directly increasingtranscription of the operatively linked gene or gene cluster compriseenhancers that can be located more distally from the transcription startsite compared to promoters, and either upstream or downstream from theregulated genes, as understood by those skilled in the art. Enhancersare typically short (50-1500 bp) regions of DNA that can be bound bytranscriptional activators to increase transcription of a particulargene. Typically, enhancers can be located up to 1 Mbp away from thegene, upstream or downstream from the start site.

In some embodiments the GVA genes and GVS genes of the GVGC hereindescribed can be provided in a polynucleotidic construct wherein all theGVA genes and the GVS genes of the GVGC cluster are under control of oneor more one or more regulatory sequences.

In other embodiments, the GVA genes and GVS genes of the GVGC hereindescribed can be provided in a plurality of polynucleotide constructs,each comprising subsets of GVA genes and/or GVS genes within operonsconfigured to allow co-expression of the GVA genes and GVS genes in oneor more prokaryotes to form a GV type.

In particular in GVGC herein described co-expression of the GVS genesand the GVA genes in connection with regulatory sequence capable ofoperating in the prokaryotic host are configured to provide a GV type.

Exemplary regulatory regions capable of operating in prokaryotescomprise promoters, enhancers, silencers, terminators, regulators,operators, ribosome binding sites, and riboswitches, among others knownin the art. Regulatory regions capable of operating in a prokaryotichost can be selected by a skilled person following selection of theprokaryotic host of interest. Exemplary constitutive and inducibleprokaryotic promoters and operators suitable for regulating expressionof GVs in a prokaryotic host comprise T7, T7lac, Sp6, araBAD, trp, lac,Ptac, pL, and others identifiable by those skilled in the art anddescribed herein. For example, the lac operator and the araBAD promoterare exemplary regulatory elements that can be used for controlling geneexpression in bacteria such as E. coli (see Example 2 and 8).

Riboswitches are another example of a regulatory sequence commonlypresent in prokaryotic untranslated regions (UTRs) of encoded RNAs.These sequences are configured to switch between alternative secondarystructures in the RNA depending on the concentration of key metabolites.The secondary structures then either block or reveal other regulatorysequence regions such as RBSs.

In some embodiments, expression of the GVGC described herein can beregulated by one or more of any native regulatory elements known in theart to control gene expression in the naturally occurring form ofprokaryotic cell. In other embodiments, a promoter regulating expressionof a GVGC can comprise regulatory elements that are regulated by afactor (e.g. a transcription factor) that is not expressed in thenaturally occurring form of the host prokaryotic cell. In someembodiments, GVGC can be regulated by inducible promoters such as thosepromoters inducible by sugars (e.g., L-arabinose, L-rhamnose, xylose andsucrose), antibiotics (e.g., tetracycline), or CRSPR-dCas9, or regulatedby heat shock promoters, pH promoters, oxidation stress promoters,radiation promoters, metal promoters, among others known in the art, orconstitutive promoters of varying strengths. In some embodiments, aheterologous factor, e.g. a polynucleotide construct encoding aheterologous transcription factor required for activation of expressionof the GVGC in the host prokaryote can be introduced into the hostprokaryote. In exemplary embodiments described herein, GVGC constructsare regulated by a bacteriophage T7 promoter and the bacteriophage T7RNA Polymerase required to activate expression of the T7 promoter isexpressed from a polynucleotide introduced into the prokaryotic cell(e.g., Examples 2 and 8).

In particular, GVGCs herein described are provided based on thesurprising finding that in a GVGC the GVA genes and proteins and not theGVS genes and proteins determine the prokaryotic host where a GV can beprovided. In a prokaryotic host GVA genes can be expressed in afunctional or non-functional form depending on the host. Inclusion ofGVA genes capable of expression in a functional form in a prokaryotichost enable production in said host of GVs formed by GVS proteins nativeto one or more prokaryotic species possibly other than the prokaryotichost. Accordingly, it is expected that introduction in a certainprokaryotic host of a GVGC cluster comprising GVA gene that can beexpressed in a functional form in the prokaryotic host, allows formationin that host of GVs formed by GVS proteins native to any prokaryoticspecies including GVs formed by a combination GVS proteins native tomore than one prokaryotic species.

Accordingly, described herein are hybrid gene clusters comprising gasvesicle assembly (GVA) genes native to a first bacterial species andcapable of being expressed in a functional form in the prokaryotic host,and one or more gas vesicle structural (GVS) genes native to one or morebacterial species, with at least one of the one or more bacterialspecies different from the first bacterial species.

The term “hybrid gene cluster” or “hybrid cluster” as used hereinindicates a cluster comprising at least two genes native to differentspecies and resulting in a cluster not natively in any organisms.

Accordingly in embodiments described herein, GV gene clusters include acombination of GVA and GVS genes which is not native in any naturallyoccurring prokaryotes.

In some embodiments, in the GVGC of the disclosure the GVA genes arenative to a GVA prokaryotic species other than the prokaryotic host andare selected to provide GVA proteins functional in the prokaryotic host.In some embodiments, the GVA prokaryotic species is the same of the hostprokaryotic species, the GVA genes are thus native to the prokaryotichost and are included in a hybrid GVGC together with at least one GVSgene native to a prokaryotic species other than the prokaryotic hosts.

A skilled person will be able to select native GVA and GVS genes in viewof a specific GV type to be formed in the prokaryotic host according toan experimental design and engineer a hybrid GV gene cluster encodingthe GV type in a configuration allowing co-expression in the prokaryotichost In particular, in some embodiments, selecting GVA and GVS genes toform a hybrid GVGC can be performed by first identifying all of the GVAgenes in the one or more operons of a GV gene cluster of the GVAprokaryote species, as herein described. Following their identification,all of the GVA genes can then be inserted into one or more expressionconstructs, in operative connection with suitable regulatory elements(e.g. promoter and other regulatory elements described herein andcapable of regulating expression in the prokaryotic host species). Insome embodiments, this is performed together initially with theinsertion into the one or more expression constructs with GVS genes fromthe GVA prokaryotic species in order to detect whether the GVA genes arecapable of being expressed in a functional form in the prokaryotic hostspecies. In other embodiments, the GVA genes are inserted into one ormore expression constructs together with GVS genes from one or moreprokaryote species that is different from the GVA prokaryote species inorder to detect whether the GVA genes are capable of being expressed ina functional form in the prokaryotic host species.

In general, the selection of GVA genes capable of being expressed in afunctional form in a host prokaryote species can be performed bydetecting expression in the prokaryotic host of one or more candidate GVgene cluster native to a prokaryotic species other than the prokaryotichost to obtain a GVA prokaryotic cell capable of forming detectable GVsin the prokaryotic host, as would be understood by a skilled person uponreading of the present disclosure.

In general, selection of promoter and other regulatory sequences to beincluded in expression polynucleotidic constructs can be performed byone or more of the following: detecting functionality of a promoterand/or additional regulatory sequence in the host cells, selectingpromoters and/or additional regulatory sequences known to be functionalin the host cells; detecting the strength of the promoters and/oradditional regulatory sequences in connection with protein productionand/or selecting promoter and/or additional regulatory sequences ofknown strength; and selecting inducible promoters and/or additionalregulatory sequence to control GV expression.

In particular some embodiments, identification and selection of GVAcapable of being expressed in a functional form in a prokaryotic hostcan be performed by detecting expression in the prokaryotic host of oneor more candidate GV gene cluster native to a prokaryotic species otherthan the prokaryotic host and repeating the detecting until formation ofa GV by at least one candidate GV gene cluster is detected.

Accordingly, the detecting expression is performed to obtain GV genecluster comprising GVA genes native to prokaryotic cell other than theprokaryotic host and capable of forming detectable GVs in theprokaryotic host.

In some embodiments, the detecting expression is preceded by introducingthe prokaryotic host the one or more candidate GV gene cluster native toa prokaryotic species other than the prokaryotic host.

The providing and introducing of the one or more expression constructscan be performed by methods identifiable by a skilled person uponreading of the present disclosure. In particular, vectors suitable fortransferring the expression constructs into prokaryotic cells such asbacteria or archaea and methods of transformation of prokaryotic cellssuch as electroporation, heat-shock transformation and others are knownin the art and described herein (e.g., see Examples). Methods ofculturing prokaryotic cells to allow analysis of gene expression and fordetermination of GV formation are known in the art and described herein.

Detection of GVs in the prokaryotic host cells can be performed forexample by analysis following a period of time to allow expression ofthe determining if the cells become buoyant after centrifugation, or bydetecting them using transmission electron microscopy and/or additionalmethods identifiable by a skilled person upon reading of the presentdisclosure (see also Example 14 and methods described in U.S.application Ser. No. 15/613,104 filed on Jun. 2, 2017 incorporatedherein by reference in its entirety).

In some embodiment, GVA genes native capable of forming detectable GVsin the prokaryotic host selected with methods herein described can beused to provide a hybrid GV gene cluster further comprising GVS genesnative to one or more prokaryotic species including at least one GVSgene native to a prokaryotic species other than the GVA prokaryoticspecies.

Following identification and selection of GVA genes capable of beingexpressed in a functional form, a hybrid GVGC can be constructed,comprised in one or more expression constructs, comprising all of theGVA genes from the GVA prokaryotic species, together with one or moreGVS genes from a prokaryotic species different to the GVA prokaryoticspecies, the GVS proteins genes selected for their structural capabilityto form GVs with suitable contrast-enhanced imaging properties, asdescribed herein (e.g. see Examples). In particular, all of the gvpA/Bgenes from one GVS prokaryotic species are inserted into the one or moreexpression constructs (e.g. both of the two gvpA genes from Anabaenafloc-aquae, see Examples). Further, optionally a gvpC or a variantthereof from either a same or a different GVS prokaryotic species as thesource of the gvpA/B genes can be inserted into the expressionconstructs (e.g. Anabaena floc-aquae gvpC, see Examples).

In some embodiments production of a hybrid gas vesicle gene cluster(GVGC) configured for expression in the prokaryotic host can beperformed providing a polynucleotidic construct comprising gas vesicleassembly (GVA) genes native to a GVA prokaryotic species and capable offorming detectable GVs in the prokaryotic host. In the method, thepolynucleotidic construct further comprises gas vesicle structural (GVS)genes native to one or more GVS prokaryotic species, at least one of theone or more prokaryotic species different from the GVA prokaryoticspecies, in a configuration allowing co-expression of the gas vesiclestructural genes and the gas vesicle assembly genes upon operativeconnection of the with regulatory sequence configured to operate in theprokaryotic host.

In some embodiments, the polynucleotidic construct comprises onepolynucleotide. For example, all of the GVA genes and GVS genes of aGVGC can be provided in one operon, operatively connected and underregulatory control of the same promoter. In exemplary described herein,a hybrid GVGC comprises GVA genes from Anabaena floc-aquae comprisingtwo Anabaena floc-aquae GvpA genes, optionally with an Anabaenafloc-aquae gvpC gene together with all of the GVA genes from B.megaterium, gvpR, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT andgvpU (e.g. see FIG. 2A).

In some embodiments the polynucleotidic construct comprises a pluralityof polynucleotides. For example, a subset of all of the GVA genes andGVS genes are comprised in one operon, operatively connected and underregulatory control of a first promoter, whereas another subset of all ofthe GVA genes and GVS genes are comprised in another operon, operativelyconnected and under regulatory control of a second promoter. An exampleof a GV gene cluster comprising two operons is in the nativeHalobacterium genome, wherein one operon comprises a subset of all ofthe Halobacterium GVA genes, and another operon comprises a secondsubset of all of the Halobacterium GVA and GVS genes (e.g. see FIG. 20).Accordingly, it is expected that a hybrid GVGC can be constructed havinga similar configuration wherein two or more operons can be provided thattogether comprise all of the gyp genes of a hybrid GVGC

In general, the selection of the regulatory elements to obtainexpression of hybrid GV types at a concentration that will allowdetection by MRI and/or ultrasound can be determined empirically.Factors to consider in selecting regulatory elements comprise thecompatibility of the regulatory elements with the prokaryotic host, thestrength of the promoter (strong vs. weak promoters are known in theart), use of alternative RBS, enhancer elements, and alternative codonusage, among others. For hybrid GVGC expression constructs comprisinginducible promoters, the concentration of and duration of presence inthe prokaryotic host of a drug or other molecule used to activate thepromoter and induce expression of the hybrid GVGC can affect theexpression level. Further, the fraction of prokaryotic cells expressinghybrid GVGCs within an imaging target site is another considerationrelevant to imaging modalities described herein. [Q: please confirm]

In exemplary embodiments described herein, to enable a broad range of invivo applications, noninvasive imaging can be performed to detectrelatively dilute cellular populations. For example, to determine thedetection limit of exemplary hybrid GVGC-expressing cells, aconcentration series of E. coli transformed with exemplary hybrid GVGC(ARG1) was imaged using ultrasound (FIG. 4 Panel A). Cells atconcentrations as low as 5×10⁷ cells/ml produced detectable signal (FIG.4, Panels A and B), equating to a 0.005% volume fraction, orapproximately 100 cells per voxel based on cubic voxel dimensions of 100μm, providing imaging sensitivity sufficient for many in vivo scenarios(see Example 2).

Similarly, in exemplary embodiments wherein MRI imaging modalities areused, in embodiments wherein the cells occupy 100% of the tissue (e.g.in a tumor or abscess full of bacteria) then it is estimated that ˜0.06%of the cell needs to be filled with GVs to become detectable.

In particular, in some embodiments described herein whereinintracellular spatial translocation of an expressed hybrid GV type is areportable endpoint of a biochemical event in a cell, selection of anexpression level of hybrid GVs suitable for imaging modalities such asultrasound and MRI (e.g. to detect clustering) can be determinedempirically.

In the method the prokaryotic host can be of a bacterial speciesdifferent from the GVA prokaryotic species and the GVA prokaryoticspecies is different from at least one of the GVS prokaryotic species.

In some embodiments, polynucleotidic constructs comprising hybrid GVGCherein described can be used in methods and systems can be provided toproduce a gas vesicle protein structure in a prokaryotic host.

The term “polynucleotide” as used herein indicates an organic polymercomposed of two or more monomers including nucleotides, or analogsthereof. The term “nucleotide” refers to any of several compounds thatconsist of a ribose (ribonucleotide) or deoxyribose(deoxyribonucleotides) sugar joined to a purine or pyrimidine base andto a phosphate group, and that are the basic structural units of nucleicacids. The term “nucleotide analog” refers to a nucleotide in which oneor more individual atoms have been replaced with a different atom with adifferent functional group. Accordingly, the term polynucleotideincludes nucleic acids of any length of DNA or RNA analogs and fragmentsthereof. A polynucleotide of three or more nucleotides is also callednucleotidic oligomers or oligonucleotide.

In those embodiments, methods comprise introducing into the prokaryotichost a hybrid gas vesicle gene cluster (GVGC) herein describedconfigured for expression in the prokaryotic host, in which the gasvesicle structural gene native to the second prokaryotic species encodefor the gas vesicle type and, and expressing the hybrid GVGC in thebacterial host to produce the gas vesicle protein structure comprisingthe GVS proteins encoded by the GVS genes of the hybrid GVGC.

The term “protein” as used herein indicates a polypeptide with aparticular secondary and tertiary structure that can interact withanother molecule and in particular, with other biomolecules includingother proteins, DNA, RNA, lipids, metabolites, hormones, chemokines,and/or small molecules. The term “polypeptide” as used herein indicatesan organic linear, circular, or branched polymer composed of two or moreamino acid monomers and/or analogs thereof. The term “polypeptide”includes amino acid polymers of any length including full-lengthproteins and peptides, as well as analogs and fragments thereof. Apolypeptide of three or more amino acids is also called a proteinoligomer, peptide, or oligopeptide. In particular, the terms “peptide”and “oligopeptide” usually indicate a polypeptide with less than 100amino acid monomers. In particular, in a protein, the polypeptideprovides the primary structure of the protein, wherein the term “primarystructure” of a protein refers to the sequence of amino acids in thepolypeptide chain covalently linked to form the polypeptide polymer. Aprotein “sequence” indicates the order of the amino acids that form theprimary structure. Covalent bonds between amino acids within the primarystructure can include peptide bonds or disulfide bonds, and additionalbonds identifiable by a skilled person. Polypeptides in the sense of thepresent disclosure are usually composed of a linear chain of alpha-aminoacid residues covalently linked by peptide bond or a synthetic covalentlinkage. The two ends of the linear polypeptide chain encompassing theterminal residues and the adjacent segment are referred to as thecarboxyl terminus (C-terminus) and the amino terminus (N-terminus) basedon the nature of the free group on each extremity. Unless otherwiseindicated, counting of residues in a polypeptide is performed from theN-terminal end (NH₂-group), which is the end where the amino group isnot involved in a peptide bond to the C-terminal end (—COOH group) whichis the end where a COOH group is not involved in a peptide bond.Proteins and polypeptides can be identified by x-ray crystallography,direct sequencing, immunoprecipitation, and a variety of other methodsas understood by a person skilled in the art. Proteins can be providedin vitro or in vivo by several methods identifiable by a skilled person.In some instances where the proteins are synthetic proteins in at leasta portion of the polymer two or more amino acid monomers and/or analogsthereof are joined through chemically-mediated condensation of anorganic acid (—COOH) and an amine (—NH₂) to form an amide bond or a“peptide” bond.

As used herein the term “amino acid”, “amino acid monomer”, or “aminoacid residue” refers to organic compounds composed of amine andcarboxylic acid functional groups, along with a side-chain specific toeach amino acid. In particular, alpha- or α-amino acid refers to organiccompounds composed of amine (—NH2) and carboxylic acid (—COOH), and aside-chain specific to each amino acid connected to an alpha carbon.Different amino acids have different side chains and have distinctivecharacteristics, such as charge, polarity, aromaticity, reductionpotential, hydrophobicity, and pKa. Amino acids can be covalently linkedto form a polymer through peptide bonds by reactions between the aminegroup of a first amino acid and the carboxylic acid group of a secondamino acid. Amino acid in the sense of the disclosure refers to any ofthe twenty naturally occurring amino acids, non-natural amino acids, andincludes both D an L optical isomers.

In particular, in embodiments herein described one or morepolynucleotidic constructs comprising a hybrid GVGC herein describedoperatively connected to regulatory sequences can be provided in aconfiguration designed for heterologous expression of any type of gasvesicle (see also U.S. application Ser. No. 15/613,104 filed on Jun. 2,2017 incorporated herein by reference in its entirety) in any type ofprokaryotic cell.

As used herein, “heterologous expression” refers to expression of GVs inany species that either does not naturally produce gas vesicles, orwhere its natural production of gas vesicles has been suppressed, forexample through genetic knock-out of the genes encoding Gvp proteins,and where foreign DNA encoding gas vesicle genes is introduced into theorganism to persist as a plasmid or integrate into the genome.

In some embodiments, heterologously expressed Gvp genes can comprisegenes encoding corresponding Gvp proteins which are naturally occurringor have sequences having at least 50% identity with naturally occurringGvp proteins.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the nucleotidebases or residues in the two sequences that are the same when alignedfor maximum correspondence over a specified comparison window. Whenpercentage of sequence identity or similarity is used in reference toproteins, it is recognized that residue positions which are notidentical often differ by conservative amino acid substitutions, whereamino acid residues are substituted with a functionally equivalentresidue of the amino acid residues with similar physiochemicalproperties and therefore do not change the functional properties of themolecule.

A functionally equivalent residue of an amino acid used herein typicallyrefers to other amino acid residues having physiochemical andstereochemical characteristics substantially similar to the originalamino acid. The physiochemical properties include water solubility(hydrophobicity or hydrophilicity), dielectric and electrochemicalproperties, physiological pH, partial charge of side chains (positive,negative or neutral) and other properties identifiable to a personskilled in the art. The stereochemical characteristics include spatialand conformational arrangement of the amino acids and their chirality.For example, glutamic acid is considered to be a functionally equivalentresidue to aspartic acid in the sense of the current disclosure.Tyrosine and tryptophan are considered as functionally equivalentresidues to phenylalanine. Arginine and lysine are considered asfunctionally equivalent residues to histidine.

A person skilled in the art would understand that similarity betweensequences is typically measured by a process that comprises the steps ofaligning the two polypeptide or polynucleotide sequences to form alignedsequences, then detecting the number of matched characters, i.e.characters similar or identical between the two aligned sequences, andcalculating the total number of matched characters divided by the totalnumber of aligned characters in each polypeptide or polynucleotidesequence, including gaps. The similarity result is expressed as apercentage of identity.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison,and multiplying the result by 100 to yield the percentage of sequenceidentity.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length protein or protein fragment. A reference sequence cancomprise, for example, a sequence identifiable a database such asGenBank and UniProt and others identifiable to those skilled in the art.

As understood by those skilled in the art, determination of percentidentity between any two sequences can be accomplished using amathematical algorithm. Non-limiting examples of such mathematicalalgorithms are the algorithm of Myers and Miller [13], the localhomology algorithm of Smith et al. [14]; the homology alignmentalgorithm of Needleman and Wunsch [15]; the search-for-similarity-methodof Pearson and Lipman [16]; the algorithm of Karlin and Altschul [17],modified as in Karlin and Altschul [18]. Computer implementations ofthese mathematical algorithms can be utilized for comparison ofsequences to determine sequence identity. Such implementations include,but are not limited to: CLUSTAL in the PC/Gene program (available fromIntelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0)and GAP, BESTFIT, BLAST, FASTA [16], and TFASTA in the WisconsinGenetics Software Package, Version 8 (available from Genetics ComputerGroup (GCG), 575 Science Drive, Madison, Wis., USA). Alignments usingthese programs can be performed using the default parameters.

In some embodiments, heterologously expressed Gvp proteins to provide aGV type have independently at least 50% sequence identity, preferably atleast 80%, more preferably at least 90%, most preferably at least 95%sequence identity compared to a reference sequence of corresponding Gvpprotein using one of the alignment programs described using standardparameters.

Heterologous expression of GVs in a prokaryotic cell can be performed bycloning one or more polynucleotides encoding naturally occurring Gvpproteins or homologs thereof that are required for production of GVs(comprising gvpA/B, gvpC, and other proteins known to those skilled inthe art and described herein) into one or more suitable constructsconfigured to express the heterologous GV proteins in the prokaryoticcell. Polynucleotides encoding GV protein genes can be cloned usingcommercially available reagents from vendors such as Qiagen, Invitrogen,Applied Biosystems, Promega, and others, following standard molecularbiology methods known in the art, such as those described herein. Aswould be understood by those skilled in the art, polynucleotidesencoding GV protein genes can be obtained from several differentsources. For example, polynucleotides encoding GV proteins can beobtained by isolating genomic DNA or cDNA encoding GV proteins frommicroorganisms whose genomes encode GV proteins genes, and/or express GVproteins RNA. RNA can be isolated from a cell that expresses GV proteinsgenes, and cDNA produced by reverse transcription using standardtechniques and commercial kits. Genomic DNA can be purified from thecell, and cDNA or genomic DNA encoding one or more GV proteins isolated,following methods known to those in the art. Alternatively,polynucleotides comprising one or more gas vesicle genes can besynthesized using oligonucleotide and polynucleotide synthetic methodsknown in the art. PCR-based amplification of one or more GV proteingenes can be performed using appropriately designed primer pairs (e.g.using PrimerDesign or other programs known to those skilled in the art).PCR-based amplification can be followed by ligation (e.g. using T4 DNAligase) of a polynucleotide encoding gas vesicle gene amplicon into anappropriate construct in a plasmid suitable for propagation in bacteriaor archea, such as transformation-competent E. coli DH5alpha, followedby growth of transformed cell cultures, purification of the plasmid forconfirmation of the cloned enzyme by DNA sequence analysis, among othermethods known to those skilled in the art. Expression vectors cancomprise plasmid DNA, viral vectors, or non-viral vectors, among othersknown to those skilled in the art, comprising appropriate regulatoryelements such as promoters, enhancers, and post-transcriptional andpost-translational regulatory sequences that are compatible with theprokaryotic cell intended to heterologously express the GV, as would beunderstood by a skilled person. In particular, in embodiments describedherein, expression vectors suitable for regulating heterologousexpression of GVs comprise those having promoters and other regulatoryelements known to skilled persons that are compatible with Gram-negativebacteria such as E. coli, and Salmonella. Promoters can beconstitutively active or inducible. Exemplary inducible expressionsystems comprise IPTG-inducible expression as shown in Examples 2 and 8.

In some embodiments, where one or more GVA and GVS proteins areexpressed heterologously to form GVs in prokaryotic cells other that thenative host, the related sequence can be optimized for expression in theheterologous host microorganism as will be understood by a skilledperson.

In particular, in some embodiments described herein, wherein a GV typeis produced heterologously in a prokaryotic cell, production of a GVgene sequences can be codon-optimized for expression in the prokaryoticcell type, for example, such as Escherichia coli, according to methodsidentifiable by a skilled person. As would be understood by thoseskilled in the art, the term “codon optimization” as used herein refersto the introduction of synonymous mutations into codons of aprotein-coding gene in order to improve protein expression in expressionsystems of a particular organism, such as E. coli in accordance with thecodon usage bias of that organism. The term “codon usage bias” refers todifferences in the frequency of occurrence of synonymous codons incoding DNA. The genetic codes of different organisms are often biasedtowards using one of the several codons that encode a same amino acidover others—thus using the one codon with, a greater frequency thanexpected by chance. Optimized codons in microorganisms, such asEscherichia coli or Salmonella typhimurium, reflect the composition oftheir respective genomic tRNA pool. The use of optimized codons can helpto achieve faster translation rates and high accuracy.

In some embodiments, many statistical methods proposed and used toanalyze codon usage bias the field of bioinformatics and computationalbiology can be used for codon optimization in the sense of thedisclosure. Methods such as the ‘frequency of optimal codons’ (Fop), theRelative Codon Adaptation (RCA) or the ‘Codon Adaptation Index’ (CAI)are used to predict gene expression levels, while methods such as the‘effective number of codons’ (Nc) and Shannon entropy from informationtheory are used to measure codon usage evenness. Multivariatestatistical methods, such as correspondence analysis and principalcomponent analysis, are widely used to analyze variations in codon usageamong genes. There are many computer programs to implement thestatistical analyses enumerated above, including CodonW, GCUA, INCA, andothers identifiable by those skilled in the art. Several softwarepackages are available online for codon optimization of gene sequences,including those offered by companies such as GenScript, EnCorBiotechnology, Integrated DNA Technologies, ThermoFisher Scientific,among others known those skilled in the art. Those packages can be usedin providing Gvp proteins with codon ensuring optimized expression invarious prokaryotic cell systems as will be understood by a skilledperson.

A representative example of heterologous GVs is the E. coli expressing aheterologous GV gene cluster from Bacillus megaterium (Mega). Mega GVsare typically cone-tipped cylindrical structures with a diameter ofapproximately 73 nm and length of 100-600 nm, encoded by a cluster ofeleven or fourteen different genes, including the primary structuralprotein, GvpB, and several putative minor components and putativechaperones [11, 19] as would be understood by a person skilled in theart. In some exemplary embodiments described herein (see Example 1),heterologous GV gene clusters comprise B. megaterium regulatory genesGvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU andstructural gene GvpB from B. megaterium.

In embodiments described herein, the GVGC gene cluster is a hybrid GVgene cluster comprising a combination of Gvp genes that are nativelyencoded in GV gene clusters from two or more different organisms. Inexemplary embodiments described herein, a hybrid GV gene clustercomprises a combination of genes from A. floc-aquae and B. megaterium(see Example 1). In particular, in exemplary embodiments, the hybrid GVgene cluster can comprise B. megaterium GVA assembly genes GvpR, GvpN,GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU and further comprisestructural GVS proteins genes from Anabaena floc-aquae such as GvpA andoptionally GvpC (see Example 1). In other exemplary embodiments, thehybrid GV gene cluster can comprise B. megaterium GVA genes GvpR, GvpN,GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU and further compriseGVS protein gene GvpA1, and GvpA2 from Bukholderia thailandensis orGvpA1, GvpA2, GvpA3 and GvpA4 from Psychromonas ingrahamii. (seeexemplary GVA and GVS in Example 9).

In other exemplary embodiments, the hybrid GV gene cluster can compriseB. megaterium GVA genes GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ,GvpT and GvpU and further comprise GVS protein genes GvpB from B.megaterium and GvpC from Anabaena floc-aquae.

Thus, in general, according to embodiments described herein, hybrid GVGCcan comprise GVS genes having GvpA/B all from one species, optionallytogether with GvpC from the same species as GvpA/B or GvpC from adifferent species. In some embodiments herein described, hybrid GVGC areintroduced in a prokaryotic cell to provide a reportable molecularcomponent of a gas vesicle reporting (GVR) genetic circuit in operativeconnection with other molecular components of the genetic circuit toreport occurrence of a biochemical event in the prokaryotic cell.

In the GVR genetic circuits in the sense of the present disclosure, themolecular components forming parts of the GVR genetic circuit can begenetic molecular components or cellular molecular components.

The term “molecular component” as used in connection with the GVRgenetic circuits described herein indicates a chemical compound or astructure comprised of a plurality of chemical compounds comprised in acellular environment. Exemplary molecular components thus comprisepolynucleotides, such as ribonucleic acids or deoxyribonucleic acids,polypeptides, polysaccharides, lipids, amino acids, peptides, sugarsand/or other small or large molecules and/or polymers that can be foundin a cellular environment. In some embodiments described herein, amolecular component of a GVR genetic circuit is a GV type or a clusterthereof.

The term “genetic molecular component” as used herein indicates amolecular unit formed by a gene (possibly comprising or formed by acluster of genes), an RNA transcribed from the gene or a portion thereofand optionally a polypeptide or a protein translated from thetranscribed RNA. In genetic circuits herein described, the biochemicalreactions connecting the genetic molecular component to anothermolecular component of the circuit can involve any one of the gene, thetranscribed RNA and/or the polypeptide forming the molecular component.

A gene comprised in a genetic molecular component is a polynucleotidethat can be transcribed to provide an RNA and typically comprises codingregions as well as one or more regulatory sequence regions which is asegment of a nucleic acid molecule which is capable of increasing ordecreasing transcription or translation of the gene within an organismeither in vitro or in vivo. In particular coding regions of a geneherein described can comprise one or more protein coding regions whichwhen transcribed and translated produce a polypeptide, or if an RNA isthe final product only a functional RNA sequence that is not meant to betranslated. Regulatory regions of a gene herein described comprisepromoters, transcription factor binding sites, operators, activatorbinding sites, repressor binding sites, enhancers, protein-proteinbinding domains, RNA binding domains, DNA binding domains, silencers,insulators and additional regulatory regions that can alter geneexpression in response to stimuli as will be recognized by a personskilled in the art.

An RNA of a genetic molecular component comprises any RNA that can betranscribed from a gene, such as a messenger ribonucleic acid (mRNA),short interfering ribonucleic acid, and ribonucleic acid capable ofacting as regulating factors in the cell. mRNA comprised in a geneticmolecular component comprise regions coding for the protein as well asregulatory regions e.g. ribosome binding site domains (“RBS”), which isa segment of the upstream (5′) part of an mRNA molecule to which theribosomal machinery of a cell binds to position the message correctlyfor the initiation of translation. RBSs control the accuracy andefficiency with which the translation of mRNA begins. mRNA can haveadditional control elements encoded, such as riboregulator sequences orother sequences that form hairpins, thereby blocking the access of theribosome to the Shine-Delgarno sequence and requiring an externalsource, such as an activating RNA, to obtain access to theShine-Delgarno sequence. Other RNAs that serve regulatory roles that cancomprise the genetic molecular component include riboswitches, aptamers(e.g. malachite green, Spinach), aptazymes, guide CRISPR RNAs, and otherRNAs known to those skilled in the art.

A protein comprised in a molecular component can be proteins withactivating, inhibiting, binding, converting, or reporting functions.Proteins that have activating or inhibiting functions typically act onoperator sites encoded on DNA, but can also act on other molecularcomponents. Proteins that have binding functions typically act on otherproteins, but can also act on other molecular components. Proteins thathave converting functions typically act on small molecules, and convertsmall molecules from one small molecule to another by conducting achemical or enzymatic reaction. Proteins with converting functions canalso act on other molecular components. Proteins with reportingfunctions have the ability to be easily detectable by commonly useddetection methods (absorbance, fluorescence, for example), or otherwisecause a reaction on another molecular component that causes easydetection by a secondary assay (e.g. adjusts the level of a metabolitethat can then be assayed for). The activating, inhibiting binding,converting, or reporting functions of a protein typically form theinteractions between genetic components of a genetic circuit. Exemplaryproteins that can be comprised in a genetic molecular component comprisemonomeric proteins and multimeric proteins, proteins with tertiary orquaternary structure, proteins with linkers, proteins with non-naturalamino acids, proteins with different binding domains, and other proteinsknown to those skilled in the art. Specific exemplary proteins includeTetR, LacI, LambdaCI, PhlF, SrpR, QacI, BetR, LmrA, AmeR, LitR, met,AraC, LasR, LuxR, IpgC, MxiE, Gal4, GCN4, GR, SP1, CREB, and othersknown to a skilled person in the art.

The term “cellular molecular component” indicates a molecular componentnot encoded by a gene, or indicates a molecular component transcribedand/or translated by a gene but comprised in the circuit without thecorresponding gene. Exemplary cellular components comprisepolynucleotides, polypeptides, polysaccharides, small molecules andadditional chemical compounds that are present in a cellular environmentand are identifiable by a skilled person. Polysaccharides, smallmolecules, and additional chemical compounds can include, for example,NAD, FAD, ATP, GTP, CTP, TTP, AMP, GMP, ADP, GDP, Vitamin B1, B12,citric acid, glucose, pyruvate, 3-phosphoglyceric acid,phosphoenolpyruvate, amino acids, PEG-8000, FiColl 400, spermidine, DTT,b-mercaptoethanol maltose, maltodextrin, fructose, HEPES, Tris-Cl,acetic acid, aTc, IPTG, 3OC12HSL, 3OC6HSL, vanillin, malachite green,Spinach, succinate, tryptophan, and others known to those skilled in theart. Polynucleotides can include RNA regulatory factors (smallactivating RNA, small interfering RNA), or “junk” decoy DNA that eithersaturates DNA-binding enzymes (such as exonuclease) or contains operatorsites to sequester activator or repressor enzymes present in the system.Polypeptides can include those present in the genetic circuit but notproduced by genetic components in the circuit, or those added to affectthe molecular components of the circuit.

In some embodiments of genetic circuits herein described, one or moremolecular components is a recombinant molecular component that can beprovided by genetic recombination (such as molecular cloning) and/orchemical synthesis to bring together molecules or related portions frommultiple sources, thus creating molecular components that would nototherwise be found in a single source.

In embodiments herein described, a genetic circuit comprises at leastone genetic molecular component or at least two genetic molecularcomponents, and possibly one or more cellular molecular components,connected one to another in accordance with a circuit design byactivating, inhibiting, binding or converting reactions to form a fullyconnected network of interacting components.

In embodiments of the GVR genetic circuits described herein, themolecular components are connected one with another according to acircuit design in which a molecular component is an input and anothermolecular component is an output. In particular, a genetic circuittypically has one or more input or start molecular component whichactivates, inhibits, binds and/or convert another molecular component,one or more output or end molecular component which are activated,inhibited, bound and/or converted by another molecular component, andintermediary molecular components each inhibiting, binding and/orconverting another molecular component and being activated, inhibited,bound and/or converted by another molecular component. In someembodiments of the genetic circuits herein described, the input is thebiochemical event and/or a trigger molecular component and the output isactivation of expression of a GV gene cluster and assembly of a GV typethrough binding reactions between Gvps of the GV type. In otherembodiments of the genetic circuits herein described, the input is abiochemical event and/or a trigger molecular component and the output isan intracellular spatial translocation of the GV type, the intracellularspatial translocation occurring typically through one or more convertingand/or binding reactions as described herein. The output of GVR circuitherein described can be detected with ultrasound contrast, MRI SWI,light scattering and additional techniques to detect GV identifiable bya skilled person upon reading of the present disclosure.

The term “activating” as used herein in connection with a molecularcomponent of a genetic circuit refers to a reaction involving themolecular component which results in an increased presence of themolecular component in the cellular environment. For example, activationof a genetic molecular component indicates one or more reactionsinvolving the gene, RNA and/or protein of the genetic molecularcomponent resulting in an increased presence of the gene, RNA and/orprotein of the genetic molecular component (e.g. by increased expressionof the gene of the molecular component, and/or an increased translationof the RNA). An example of “activating” described herein comprises theinitiation of expression of a GV gene cluster regulated by anIPTG-inducible promoter (e.g., see Examples 2 and 8).

Activation of a molecular component of a genetic circuit by anothermolecular component of the circuit can be performed by direct orindirect reaction of the molecular components. Examples of a directactivation of a genetic molecular component comprise in a circuit theproduction of an alternate sigma factor (molecular component of thecircuit) that drives the expression of a gene controlled by thealternate sigma factor promoter (other molecular component of thecircuit), or the production of a small ribonucleic acid (molecularcomponent of the circuit) that increases expression of ariboregulator-controlled RNA (molecular component of the circuit).Examples of indirect activation of a genetic molecular componentcomprise the production of a first protein that inhibits an intermediatetranscriptional repressor protein, wherein the intermediatetranscriptional repressor protein represses the production of a targetgene, such that the first protein indirectly activates expression of thetarget gene.

The term “inhibiting” as used herein in connection with a molecularcomponent of a genetic circuit refers to a reaction involving themolecular component of the genetic circuit and resulting in a decreasedpresence of the molecular component in the cellular environment. Forexample, inhibition of a genetic molecular component indicates one ormore reactions involving the gene, RNA and/or protein of the geneticmolecular component resulting in a decreased presence of the gene, RNAand/or protein (e.g. by decreased expression of the gene of themolecular component, and/or a decreased translation of the RNA).Inhibition of a cellular molecular component indicates one or morereactions resulting in a decreased production or increased conversion,sequestration or degradation of the cellular molecular components (e.g.a polysaccharide or a metabolite) in the cellular environment.

Inhibition can be performed in the genetic circuit by direct reaction ofa molecular component of the genetic circuit with another molecularcomponent of the circuit or indirectly by reaction of products of areaction of the molecular components of the genetic circuit with theanother molecular component of the circuit.

The term “binding” as used herein in connection with molecularcomponents of a genetic circuit refers to the connecting or uniting twoor more molecular components of the circuit by a bond, link, force ortie in order to keep two or more molecular components together, whichencompasses either direct or indirect binding where, for example, afirst molecular component is directly bound to a second molecularcomponent, or one or more intermediate molecules are disposed betweenthe first molecular component and the second molecular component anothermolecular component of the circuit. Exemplary bonds comprise covalentbond, ionic bond, van der Waals interactions and other bondsidentifiable by a skilled person.

In some embodiments, the binding can be direct, such as the productionof a polypeptide scaffold that directly binds to a scaffold-bindingelement of a protein. In other embodiments, the binding may be indirect,such as the co-localization of multiple protein elements on onescaffold. In some instances binding of a molecular component withanother molecular component can result in sequestering the molecularcomponent, thus providing a type of inhibition of said molecularcomponent. In some instances binding of a molecular component withanother molecular component can change the activity or function of themolecular component, as in the case of allosteric interactions betweenproteins, thus providing a type of activation or inhibition of the boundcomponent.

The term “converting” as used herein in connection with a molecularcomponent of the circuit refers to the direct or indirect conversion ofthe molecular component into another molecular component. An example ofthis is the conversion of chemical X by protein A to chemical Y that isthen further converted by protein B to chemical Z.

In the GVR genetic circuits in the sense of the present disclosure, theGVGC genes are introduced into a prokaryotic cell to provide areportable molecular component connected with other genetic or cellularmolecular components according to a circuit design, wherein the GV typeis expressed or the GV type is intracellularly spatially translocatedwhen the GVGC genetic circuit operates according to the circuit designin response to a biochemical event and/or to a trigger molecularcomponent.

The term “reportable molecular component” as used herein indicates amolecular component capable of detection in one or more systems and/orenvironments. The terms “detect” or “detection” as used herein indicatesthe determination of the existence, presence or fact of a target in alimited portion of space, including but not limited to a sample, areaction mixture, a molecular complex and a substrate. The “detect” or“detection” as used herein can comprise determination of chemical and/orbiological properties of the target, comprising ability to interact, andin particular bind other compounds, ability to activate another compoundand additional properties identifiable by a skilled person upon readingof the present disclosure. The detection can be quantitative orqualitative. A detection is “quantitative” when it refers, relates to,or involves the measurement of quantity or amount of the target orsignal (also referred as quantitation), which includes but is notlimited to any analysis designed to determine the amounts or proportionsof the target or signal. A detection is “qualitative” when it refers,relates to, or involves identification of a quality or kind of thetarget or signal in terms of relative abundance to another target orsignal, which is not quantified. In particular, in embodiments hereindescribed detection of the reportable molecular component comprising aGV type is performed through contrast enhanced imaging techniques suchas ultrasound and MRI.

The term “biochemical event” as used herein refers to an activating,inhibiting, binding or converting reaction between two or more molecularcomponents within a prokaryotic cell.

Accordingly, in some embodiments, at least one genetic molecularcomponent of the GVR genetic circuit comprises a hybrid gas vesicle (GV)gene cluster operatively connected to a promoter configured to beactivated directly or indirectly by the biochemical event, and directlyinitiate expression of a GV type.

In some embodiments herein described, a genetic molecular component ofthe GVR genetic circuit comprises a gas vesicle (GV) gene cluster or oneor more genes thereof operatively connected to one or more promotersconfigured to be activated directly or indirectly by the biochemicalevent, and directly initiate expression of a GV type. In someembodiments, the GVGC is comprised in a genetically engineeredpolynucleotide construct optionally comprising one or more enhancersand/or other regulatory DNA elements identifiable by those skilled inthe art. As would be understood by those skilled in the art, promotersare DNA regulatory elements that are typically located adjacent to thetranscription start sites of genes, or a cluster of genes, on the samestrand and upstream on a DNA sequence (towards the 5′ region of thesense strand), and for transcription to occur, the enzyme thatsynthesizes RNA, known as RNA polymerase, attaches to the promoter.Promoters contain DNA sequences identifiable by those skilled in theart, such as those that provide binding sites for RNA polymerase andalso for proteins that function as transcription regulatory factors thatcan either activate or repress gene transcription.

The term “transcription regulatory factor” or “transcription factor” asused herein refers to any type of factors that can function by acting ona regulatory DNA element such as a promoter or enhancer sequence. Thetranscription regulatory factors can be broadly classified into atranscription repression factor (also referred to as “repressor”) and atranscription activation factor (also referred to as “activator”). Thetranscription repression factor acts on a regulatory DNA element torepress the transcription of a gene, thereby reducing the expressionlevel of the gene. The transcription activation factor acts on aregulatory DNA element to promote the transcription of a gene, therebyincreasing the expression level of the gene.

In particular, a transcription regulatory factor has typically at leastone DNA-binding domain that can bind to a specific sequence of enhanceror promoter sequences. Some transcription factors bind to a DNA promotersequence near the transcription start site and help form thetranscription initiation complex. Other transcription factors bind toother regulatory sequences, such as enhancer sequences, and can eitherstimulate or repress transcription of the related gene.

Examples of specific transcription repression factors include TetR,LacI, LambdaCI, PhlF, SrpR, QacI, BetR, LmrA, AmeR, LitR, met, and otheridentifiable by a skilled person, as well as homologues of knownrepression factors, that function in both prokarayotic and eukarayoticsystems. Examples of transcription activation factors include AraC,LasR, LuxR, IpgC, MxiE, Gal4, GCN4, GR, SP1, CREB, etc as well ashomologues of known activation factors, that function in prokarayoticsystems.

In some embodiments, one or more promoters operatively connected to oneor more genes of a GV gene cluster is configured to be activateddirectly or indirectly by one or more biochemical events. In particular,in some embodiments, activation of expression of a GV gene cluster canbe linked to another molecular component in the GVR genetic circuitthrough activator or repressor transcription factors. In someembodiments, expression of the transcription factors can be regulated bya promoter of interest (see Example 2 and 8). In other embodiments,transcription factors can be regulated post-translationally throughdegradation or phosphorylation of the transcription factor.

Accordingly, the reportable genetic molecular component of the GVRgenetic circuit comprising a gas vesicle (GV) gene cluster operativelyconnected to a promoter configured to be activated directly orindirectly by the biochemical event, and directly initiate expression ofa GV type can in several embodiments comprise promoters and/or other DNAregulatory elements having one or more sequences identifiable to thoseskilled in the art that are configured to function as binding sites forany known transcription regulatory factor.

For example, in some embodiments GVGC expression can be activated bypromoters inducible by sugars (e.g., L-arabinose, L-rhamnose, xylose andsucrose), antibiotics (e.g., tetracycline), CRISPR-dCas9, heat shockpromoters, pH-dependent promoters, oxidation stress-dependent promoters,radiation-dependent promoters, metal-inducible promoters, and othersidentifiable by those skilled in the art. In other embodiments GVGCexpression can be induced by activation of constitutive promoters ofvarying strengths that are suitable for regulating expression inbacterial cells described herein and identifiable by those skilled inthe art.

In other embodiments, the hybrid GV gene cluster or one or more of theregulatory elements is surrounded by recombination sites that arerecognized by a recombinase, whose expression or activity is connectedthrough the genetic circuit to a biochemical event in the bacterialcell. For example, a GV gene cluster in reverse (3′-5′) orientation toits promoter (in 5′-3′ orientation) can be flanked by recombinationsites surrounding the hybrid GV gene cluster, the recombination sitesconfigured to allow inversion of the hybrid GV gene cluster uponexpression or activation of its respective recombinase, wherein uponrecombination the hybrid GV gene cluster is flipped into a 5′-3′orientation to allow initiation of expression by the promoter. Suitablerecombination systems for use in bacteria are identifiable by thoseskilled in the art, such as the Flp-FRT recombination system.

In embodiments described herein, a GV gene cluster comprised in one ormore genetic molecular components of the GVR genetic circuits describedherein is configured to function as a set of reporter genes, whichtogether encode proteins required for the formation of a GV type, suchthat expression of the GV type functions as a genetically-encodedreporter of the biochemical event in the prokaryotic cell comprising aGVR genetic circuit. As described herein, the reportable characteristicsof the GV are such that the genetically-encoded GV can be used as acontrast agent, which, when used together with one or morecontrast-enhanced imaging techniques described herein, functions as agenetically-encoded reporter in prokaryotic cells that have beengenetically engineered to comprise one or more of the GVR geneticcircuits described herein.

In particular, in exemplary embodiments described herein, all the GVAgenes GvpF, GvpG, GvpJ, GvpL, GvpK, GvpS, and GvpU and GVS gene GvpAenable GV formation. Therefore, if expression any one of these genes isregulated according to the design of a GVR genetic circuit as describedherein then the expression of the GV type will be regulated accordingly.

In some embodiments, the GVR genetic circuits described herein cancomprise a plurality of genetic molecular components that function asBoolean logical operators in genetic circuit designs known to thoseskilled in the art, such as those described in [20, 21]. As would beunderstood by persons skilled in the art, Boolean logic is a branch ofalgebra in which the values of the variables are the truth values ‘true’and ‘false’, usually denoted by the digital logic terms ‘1’ and ‘0’respectively. In contrast with elementary algebra where the values ofthe variables are numbers, and the main operations are addition andmultiplication, the main operations of Boolean logic are the conjunction‘AND’, the disjunction ‘OR’, and the negation ‘NOT’. As understood bythose skilled in the art, it is thus a formalism for describing logicalrelations in the same way that ordinary algebra describes numericrelations.

Accordingly, the term “AND gate” refers to a digital logic gate thatbehaves according to the truth table shown in Table 1. A ‘true’ output(1) results only if both the inputs to the AND gate are ‘true’ (1). Ifneither or only one input to the AND gate is ‘true’ (1), a ‘false’ (0)output results. Therefore, the output is always 0 except when all theinputs are 1.

TABLE 1 ‘AND gate’ truth table: Input Output A B A AND B 0 0 0 0 1 0 1 00 1 1 1

In particular, the term “AND gate” as used herein refers to the logicalrelation between two genetic molecular components in a GVR geneticcircuit, wherein inputs ‘A’ and ‘B’ in Table 1 are two biochemicalevents, and the output ‘A AND B’ in Table 1 is the GV type.

For example, in some embodiments of an “AND gate” comprised in a GVRgenetic circuit described herein, the GVR genetic circuit comprises aplurality of genetic molecular components wherein at least a firstgenetic molecular component comprises a first subset of genes from theGV gene cluster, and at least a second genetic molecular componentcomprises a second subset of genes from the GV gene cluster, whereintogether the GV proteins expressed from the first and second geneticmolecular components are configured to form a GV type. In theseembodiments, activation of both the first AND second genetic molecularcomponent is required for the output of the GV type in the geneticcircuit when the genetic circuit operates according to the design of thegenetic circuit. For example, the first and second genetic molecularcomponents can comprise promoters that are activated by two or morebiochemical events in the porkaryotic cell comprising the GVR geneticcircuit.

In exemplary embodiments, any of GVA genes GvpF, GvpG, GvpJ, GvpL, GvpK,GvpS, and GvpU and GVS gene GvpA of a GV gene cluster can be split intoat least a first and second genetic molecular component comprising atleast a first and a second subset of these genes to form an AND gate.

In other embodiments of an “AND gate” comprised in a GVGC geneticcircuit, two or more regulatory elements operatively connected to a GVgene cluster comprised in a genetic molecular component of a GVGCgenetic circuit that is activated by biochemical events A AND B wouldresult in the output of the GV type in the GVGC genetic circuit. Forexample, the promoter requires binding of two transcriptional activatorsfor activation of the promoter. In Examples described herein (see theMethods section of the Examples), GV gene clusters of exemplary ARG1 andARG2 and A2C constructs is driven by the T7 promoter that has a lacoperator downstream the promoter. The T7 RNA Polymerase is regulated bythe araBAD promoter (inducible by L-arabinose). Lad is controlled by theLad promoter (IPTG inducible). Therefore only under conditions whereinboth IPTG AND L-ara are present are GVs expressed.

The term “OR gate” refers to a digital logic gate that behaves accordingto the truth table shown in Table 2. A ‘true’ output (1) results ifeither of the inputs to the OR gate are ‘true’ (1).

TABLE 2 ‘OR gate’ truth table: Input Output A B A OR B 0 0 0 0 1 1 1 0 11 1 1

In particular, the term “OR gate” as used herein refers to the logicalrelation between two genetic molecular components in a GVGC geneticcircuit, wherein inputs ‘A’ and 13′ in Table 2 are two biochemicalevents, and the output ‘A OR B’ in Table 2 is the GV type.

For example, in some embodiments of an “OR gate” comprised in a GVGCgenetic circuit described herein, a promoter operatively connected to aGV gene cluster comprised in a genetic molecular component of a GVGCgenetic circuit that is activated by biochemical events A OR B wouldresult in the output of the GV type in the GVGC genetic circuit. Forexample, the promoter is activated by binding of either of two differenttranscriptional activators.

In other embodiments, an OR gate can be achieved through the use of twoconsecutive promoters. In exemplary embodiments, both these promoterscan be located directly upstream of the GV gene cluster or they can beindependently located directly upstream of any one or more of GVA genesGvpF, GvpG, GvpJ, GvpL, GvpK, GvpS, or GvpU and GVS gene GvpA.

In other embodiments, the GVR gene cluster or one or more of the GV genecluster regulatory elements can be flanked by recombination sites thatare recognized by a recombinase, whose expression or activity is, inturn, activated in response to a biochemical event in the bacterialcell. For example, in these embodiments, one input signal can activatethe GV gene cluster while a constitutive promoter is positioned in theopposite direction of the gene cluster. The second input would drive arecombinase that flips the promoter so that GV genes can be expressed.Exemplary recombinase systems comprise Flp-FRT recombination systemsystems and others known to those skilled in the art

The term “Negated AND gate” or “NOT gate” refers to a digital logic gatethat behaves according to the truth table shown in Table 3. A ‘true’output (1) results if either of the inputs to the OR gate are ‘true’(1).

TABLE 3 ‘Negated AND gate’ or “NOT gate” truth table: Input Output A B ANOT B 0 0 0 0 1 0 1 0 1 1 1 0

In particular, the term “Negated AND gate” or “NOT gate” as used hereinrefers to the logical relation between two genetic molecular componentsin a GVGC genetic circuit, wherein inputs ‘A’ and ‘B’ in Table 3 are twobiochemical events, and the output ‘A OR B’ in Table 3 is the GV type.

For example, in some embodiments of an “Negated AND gate” or a “NOTgate” comprised in a GVGC genetic circuit described herein, the GVGCgenetic circuit comprises a plurality of genetic molecular componentswherein at least a first genetic molecular component comprises a GV genecluster, and at least a second genetic molecular component comprises anCRISPR/Cas9 complex configured to inhibit expression of a gyp genecomprised in the GV gene cluster, e.g. a gvpA. In these embodiments,activation of expression and the first genetic molecular component andabsence of activation (or repression) of the second genetic molecularcomponent are both required for the output of a GV type in the geneticcircuit when the genetic circuit operates according to the design of thegenetic circuit. For example, the first and second genetic molecularcomponents can comprise promoters that are activated or repressed by oneor more biochemical events in the prokaryotic cell comprising the GVGCgenetic circuit.

In embodiments of the genetic circuits herein described wherein theinput is a biochemical event and the output is an intracellular spatialtranslocation of the GV type, the GV type is a molecular component ofthe genetic circuit and intracellular spatial translocation of the GVtype can occur through one or more converting and/or binding reactionsinvolving the GV type as described herein.

Exemplary binding or converting reactions that can result in anintracellular spatial translocation of the GV type comprise the use of afunctionalized, tagged hybrid GV type that is configured to bind to anintracellular compartment, or to allow clustering of the GV type.

For example, in some embodiments, hybrid GVs comprising variants of GvpCthat are fused with the kinase inducible domain of CREB will clusterupon phosphorylation of this domain by Protein Kinase A, when the cellalso contains a multimeric protein (such as ferritin) that is fused tothe KIX domain of the CREB binding protein. In another example, GvpC canbe fused with a calmodulin-binding domain such as the M13 peptide, andwill cluster upon an elevation of calcium concentration when the cellalso contains a multimeric protein such as ferritin that is fused tocalmodulin.

In those embodiments, the intracellular spatial translocation of the GVtype in the bacterial cell is a reportable output of the GVR geneticcircuit. In particular embodiments, the spatial translocation of the GVtype in the bacterial cell can be detected as a change in thecontrast-enhanced image of the target site comprising the geneticallyengineered bacterial cell expressing the GV type.

Thus, heterologously expressed GVs can also be used as dynamic sensorsfor imaging specific biochemical events in bacteria. In response tomolecular signals of interest, GVs can be designed to aggregate or formclusters in bacteria, thus leading to an increase or decrease in T2 orT2* contrast, as described herein. Differential MRI contrast based onclustering can then be produced.

In particular, the GV type expressed in the bacterial cell can include aspecific functional tag attached to a surface of the gas vesicles, forexample through genetically engineering a gvpC protein to comprise atag, as described herein. In some embodiments, the tag can be configuredto specifically bind to a corresponding tag on another GV or to a targetmolecule or sub-cellular compartment in a bacterial cell. The bondsformed between gas vesicles or between gas vesicles and the target inthe bacterial cell can include covalent bonds and non-covalentinteractions, such as ionic bonds, hydrophobic interactions, hydrogenbonds, van der Walls forces, dipole-dipole interactions and others knownto a person skilled in the art.

In some embodiments, the affinity between one binding moiety and itscorresponding moiety to which the binding moiety specifically binds canbe characterized by a dissociation constant K_(d). In some instances,K_(d) has a value less than 10⁻⁵ mol/L, or less than 10⁻⁷ mol/L. In somecases, Kd of a pair of binding moiety can be on the order of about 10⁻¹⁴mol/L. “Affinity” refers to the strength of binding, increased bindingaffinity being correlated with a lower K_(d).

In some embodiments, in the GVR genetic circuit herein described, anexpression of the GV type or an intracellular spatial translocation ofthe GV type occurs when the hybrid GVR genetic circuit operatesaccording to the circuit design in response to a trigger molecularcomponent within the target prokaryotic host;

In some embodiments, the trigger molecular component is a molecularcomponent that is capable of being natively produced in the targetprokaryotic host in its naturally occurring form. In particular, thenatively produced molecular component can be a genetic molecularcomponent or a cellular molecular component.

Examples of natively produced genetic molecular component can be one ormore RNA or protein natively encoded in the genome of the naturallyoccurring form of the prokaryote host and natively expressed by thetarget prokaryotic host. Examples of cellular molecular componentsnatively produced by the target prokaryotic host comprise metabolites ofenzymatic reactions produced by enzymes that are natively expressed bythe target prokaryotic host in its naturally occurring form.

In these embodiments, the GVR genetic circuit comprises a hybrid GV typewhen the GVR genetic circuit operates according to a circuit design inresponse to the presence of the natively produced molecular component inthe target prokaryotic cell.

In particular, in these embodiments, expression of the GVR in theprokaryotic host does not require the introduction into the host of anygenetic molecular components in addition to the genetic molecularcomponents comprising the GVGC. In these embodiments, the promoteroperatively connected to a hybrid GV gene cluster in the GVGC geneticmolecular component is configured to be activated in response tomolecular components capable of being natively produced by theprokaryotic host in its naturally occurring form, such as nativelyexpressed transcription factors. Natively produced proteins or RNAsnatively encoded in the genome of a particular host prokaryote, e.g. E.coli are identifiable by those skilled in the art, as are metabolitesproduced in biochemical reactions produced in the naturally occurringform of the prokaryotic host.

Thus, in these embodiments, the target prokaryotic host is labeled withexpression of a GV type, wherein expression of the GV type occurs inpresence of the trigger molecular component that is capable of beingnatively produced in the target prokaryotic host in its naturallyoccurring form. In several embodiments described herein, one or more GVRgenetic circuits can be introduced into a prokaryotic cell or one ormore prokaryotic cell types according to genetic engineering methodsdescribed herein and known to those skilled in the art.

In other embodiments, the trigger molecular component is a heterologousmolecular component that is not capable of being natively produced inthe target prokaryotic host in its naturally occurring form. In theseembodiments, the GVGC genetic molecular component is not configured toexpress the GV type in presence of a molecular component that is capableof being natively produced in the target prokaryotic host in itsnaturally occurring form, but is instead configured to express the GVtype in presence of one or more heterologous (non-natively produced)trigger molecular components.

In these embodiments, the trigger molecular component can be one or moreheterologous molecular components comprising a heterologous geneticmolecular component and/or a heterologous cellular molecular component.

In some embodiments, the heterologous genetic molecular component cancomprise one or more protein- and/or RNA-encoding genes and/orregulatory elements such as promoters and/or enhancer elements that arenot native to the target prokaryotic genome. In some embodiments, theheterologous genetic molecular component can be introduced into thetarget prokaryotic host in addition to the one or more genetic molecularcomponents comprising the hybrid GVGC. The additional heterologousgenetic molecular component can be a constitutively expressed or aninducible genetic molecular component.

In some embodiments, the heterologous cellular molecular component cancomprise a molecular component that is naturally present in theenvironment comprising the target prokaryotic cell, such as a metaboliteproduced by a mammalian host comprising the target prokaryotic hostcell, or it can be a molecular component that is not naturally presentin the environment comprising the target prokaryotic host cell, andintroduced into the prokaryotic host cell, such as a drug configured toactivate expression of the heterologous genetic component.

In some embodiments, the hybrid GVGC genetic molecular componentcomprises promoter and/or enhancer elements that are configured to beactivated in response to the presence of a heterologous molecularcomponent. In exemplary embodiments, the promoter is drug-induciblepromoter, such as an IPTG-inducible promoter, and activation of thepromoter and initiation of expression of the GV type occurs in presenceof the drug e.g. IPTG (e.g., see Example 2 and 8). In other exemplaryembodiments, the promoter is activated by a heterologous transcriptionfactor that is encoded in a heterologous genetic molecular componentintroduced into the target prokaryotic host in addition to the GVGCgenetic molecular component; in exemplary embodiments described herein,the GVGC genetic molecular component comprises a T7 promoter and anadditional genetic molecular component introduced into the targetprokaryotic host comprises a T7 RNA polymerase (e.g., see Example 2 and8).

In some embodiments, the GVGC genetic molecular component comprisesrecombination sites (e.g. Flp-FRT recombination sites) surrounding oneor more Gvp genes comprised in the hybrid GV gene cluster or one or moreregulatory elements (e.g. promoter) wherein the one or more Gvp genes orregulatory elements are introduced into a prokaryotic host cell in anorientation that prevents expression of the encoded GV type, e.g., thepromoter is in reverse orientation relative to the GV gene cluster; inthese embodiments a heterologous genetic molecular component comprisingthe recombinase enzymes required for flipping the orientation of theelements flanked by the recombinase sites in the GVGC genetic molecularcomponent is also introduced into the prokaryotic host cell andexpression of the GV type occurs upon recombinase-mediated flipping ofthe flanked elements in the GVGC genetic molecular component into anorientation allowing initiation of expression of the GV type.

In these embodiments, the GVR genetic circuit comprises a hybrid GV typeis when the GVR genetic circuit operates according to a circuit designin response to the presence of the one or more heterologous molecularcomponents in the target prokaryotic cell.

Thus, in these embodiments, the target prokaryotic host is labeled withexpression of a GV type, wherein expression of the GV type occurs inpresence of the heterologous trigger molecular component introduced intothe target prokaryotic host.

Accordingly, in some embodiments, a method to provide a geneticallyengineered prokaryotic cell comprising one or more GVR genetic circuitsis described. The method comprises genetically engineering a prokaryoticcell by introducing into the cell one or more GVR genetic circuitsdescribed herein.

The prokaryotic cells described herein can be genetically engineeredusing methods known to those skilled in the art. For example, one ormore genetic molecular components of a GVR genetic circuit comprised invectors described herein can be introduced into bacterial cells usingbacterial transformation techniques such as electroporation, heat shock,and others known to those skilled in the art and described herein. Insome embodiments, the genetic molecular components of a GVR geneticcircuit are introduced into the prokaryotic cell to persist as a plasmidor integrate into the genome, following methods known in the art anddescribed herein.

In some embodiments the prokaryotic cells are gram negative bacteria andin particular E. coli, Nissle 1997, and Salmonella. In some embodimentsthe prokaryotic cell can be a cyanobacteria such as Anabaena. In someembodiments, the prokaryotic cells are archea and in particularHalobacterium

In embodiments herein described, the gas vesicle reporter genes (GVGC),and related genetic circuits, vectors, genetically engineeredprokaryotic cells, compositions, methods and systems, which in severalembodiments can be used together with contrast-enhanced imagingtechniques to detect and report a biological event the location ofand/or biochemical events in genetically engineered prokaryotic cells inan imaging target site.

The term “contrast enhanced imaging” or “imaging”, as used hereinindicates a visualization of a target site performed with the aid of acontrast agent present in the target site, wherein the contrast agent isconfigured to improve the visibility of structures or fluids by devicesprocess and techniques suitable to provide a visual representation of atarget site. Accordingly a contrast agent is a substance that enhancesthe contrast of structures or fluids within the target site, producing ahigher contrast image for evaluation. In particular, as used herein, theterm “contrast agent” refers to GVs expressed in prokaryotic cellscomprised in the target site, the GVs comprised in GVGC genetic circuitsin the prokaryotic cells when the GVGC genetic circuit operatesaccording to a circuit design in response to a biochemical event, asdescribed herein.

The term “target site” as used herein indicates an environmentcomprising one or more targets intended as a combination of structuresand fluids to be contrasted, such as cells. In particular the term“target site” refers to biological environments such as cells, tissues,organs in vitro in vivo or ex vivo that contain at least one target. Atarget is a portion of the target site to be contrasted against thebackground (e.g. surrounding matter) of the target site. Accordingly, asused herein a target comprises one or more prokaryotic cells geneticallyengineered to comprise one or more GVGC genetic circuits as describedherein within any suitable environment in vitro, in vivo or ex vivo aswill be understood by a skilled person. Exemplary target sites includecollections of microorganisms, including, bacteria or archaea in asolution or other medium in vitro, as well as cells grown in an in vitroculture, including, primary mammalian, cells, immortalized cell lines,tumor cells, stem cells, and the like. Additional exemplary target sitesinclude tissues and organs in an ex vivo culture and tissue, organs, ororgan systems in a subject, for example, lungs, brain, kidney, liver,heart, the central nervous system, the peripheral nervous system, thegastrointestinal system, the circulatory system, the immune system, theskeletal system, the sensory system, within a body of an individual andadditional environments identifiable by a skilled person. The term“individual” or “subject” or “patient” as used herein in the context ofimaging includes a single plant, fungus or animal and in particularhigher plants or animals and in particular vertebrates such as mammalsand more particularly human beings.

In some embodiments herein described, the contrast enhanced imaging of atarget site is performed by imaging the target site with magneticresonance imaging (MRI).

The term “magnetic resonance imaging” or “MRI” as used herein indicatesan imaging technique performed by applying a magnetic field to a targetsite and detecting the resulting magnetic resonance. In MRI, a targetsite is positioned within a magnet provided by an MRI scanner where themagnetic field is used to align the magnetization of some atomic nucleiin the target site, and radio frequency magnetic fields are applied tosystematically alter the alignment of this magnetization. This causesthe nuclei to produce a rotating magnetic field detectable by thescanner, and this information is recorded to construct an image of thescanned area of the target site. The magnetic resonance of the targetsite is then detected, and the resulting data is analyzed to produce animage. MRI is thus performed based on nuclear magnetic resonance (NMR)property of nuclei of atoms inside the target site. For example, MRI iscommonly used in radiology to visualize a target site formed by internalstructures of the body of an individual. In this example, MRI makes useof the property of nuclear magnetic resonance (NMR) to image nuclei ofatoms inside the body.

Exemplary MRI systems comprise systems operating at around 1.5 Tesla(T), as well as commercial system which can run between 0.2 and 7 T andother systems identifiable by a skilled person.

In contrast enhanced imaging performed by MRI, an image contrast can befurther enhanced by weighting. Two forms of weighting are T1 and T2. T1,also known as spin-lattice weighting, allow magnetization to recoverbefore the magnetic resonance signal is measured by changing therepetition time. The repetition time is the time, measured inmilliseconds, from the application of an excitation pulse to theapplication of the next pulse, which shows how much of the longitudinalmagnetization recovers between each pulse. T2, or spin-spin weighting,allows magnetization to decay before the magnetic resonance signal ismeasured by changing the echo time. The echo time refers to time,measured in milliseconds, between the application of radiofrequencyexcitation pulse and the peak of the signal induced in the coil. Thespin-spin weighting rate can also be denoted T2*. T2* can be consideredan “observed” or “effective” T2 (which includes effects from themagnetic field inhomogeneity), whereas the first T2 can be consideredthe “natural” or “true” T2 of the tissue being imaged (i.e. purelyspin-spin interaction). T2* is less than or equal to T2.

In contrast enhanced imaging performed by MRI, an image contrast can befurther enhanced by quantitative susceptibility mapping (QSM) whichutilizes phase images to generate a 3D susceptibility distribution. Themapping can be performed by various techniques (COSMOS, MEDI, TKD,etc.), but ultimately the result is a calculated determination of theunderlying susceptibility value at each pixel/voxel of the image. Thesusceptibility is theoretically linearly proportional to theconcentration of the contrast material (in this case, air). Differentcontrast agent with different volumes would, therefore, producedifferent delta susceptibility per contrast agent.

Another MRI technique is Chemical Exchange Saturation Transfer (CEST).CEST works by having exchangeable solute protons that resonate at afrequency different from the bulk water protons when selectivelysaturated using RF irradiation. “Hyper-CEST” refers to a CEST techniquethat utilizes hyperpolarized agents, such as 129Xe. Using Hyper-CESTwith Xe based contrast allows imaging at much lower concentrations (i.e.increased sensitivity) compared to usual susceptibility-based MRItechniques (e.g. T1, T2/T2*, QSM). However, unlike T2/T2* and QSM,multiparametric GV multiplexing is not available in Hyper-CEST, sincethe Hyper-CEST cannot measure T2/T2* and QSM of the surrounding nuclearspin. Since Hyper-CEST requires xenon as the contrast agent gas, GVsneed to be exposed to xenon gas before acting as a Hyper-CEST contrastagent.

In contrast enhanced imaging performed by MRI, various contrast agentcan be used as it will be understood by a skilled person. In particular,existing contrast agents for MRI are primarily based on heavy metalchelates [22], superparamagnetic iron oxides and in particularsuperparamagnetic iron oxide nanoparticles (SPIONs) [23, 24],metalloproteins [25-28], molecules with chemically exchangeable nuclei[29-32] and fluorinated compounds [33]. More particularly, commonly usedcontrast agents for MRI are chelates of gadolinium, and iodinatedagents, as well as SPIONs used as conventional T2 and T2* contrastagents used in MRI applications such as in vivo cell tracking [34] [35].[36-39]. Further contrast agents are CEST agents with distinct chemicalshifts for exchanging nuclei [40, 41], and contrast agents to be used asdynamic sensors capable of imaging specific biological activities suchas neurotransmission or enzymatic function [27, 42-45], e.g.superparamagnetic structures designed to cluster in response molecularsignals of interest leading to an increase or decrease in T2 or T2*contrast [42, 44, 46].

In certain embodiments, imaging the target site comprises applying anexternal magnetic field to the target site in the subject, transmittinga radio frequency (RF) signal from a transmitter to the target site, andreceiving MRI data at a receiver. The MRI data can be analyzed using aprocessor, such as a processor configured to analyze the MRI data andproduce an MRI image from the MRI data. In certain embodiments, the MRIdata detected by the receiver includes an MRI signal (e.g., a radiofrequency MRI signal of the target site of the subject). In certainembodiments, the method includes obtaining a MRI data (e.g., signal) ofthe target site, and analyzing the MRI data (e.g., signal) to produce anMRI image of the target site. The MRI data (e.g., signal) can beobtained using a standard MRI device, or can be obtained using an MRIdevice configured to specifically detect the contrast agent used.Obtaining the MRI data (e.g., signal) can include detecting the MRI data(e.g., signal) with an MRI detector.

In certain embodiments, MRI data is obtained by applying to a subject astrong static magnetic field, a rapidly switching gradient field forspatial coding, and RF pulses with frequency matched such that the RFpulses trigger magnetic resonance signals from excited atomic nuclei atthe target site. For example, an atomic nucleus can produce magneticresonance signals when the RF pulse has a frequency that matches theresonance frequency (measured in chemical shifts (δ) in parts permillion (ppm)) of the atomic nucleus. In such cases, the nucleus absorbsthe RF pulse energy to become excited, and releases a magnetic resonancesignal when the excited nucleus subsequently relaxes to an unexcitedstate after characteristic time periods. The magnetic resonance signalsare detected by RF receiving antennas and digitized to generate the MRIdata. The MRI data is analyzed using any known method of analyzing MRIdata. In certain instances, the MRI data is analyzed to reconstruct theMRI image. For example, the MRI image is reconstructed from the MRI databy decoding the spatial information encoded in the MRI data using alinear reconstruction algorithm, such as Fourier transformation.

Additional methods to perform imaging of one or more GV types throughMRI detection alone or in combination with ultrasound which areapplicable in the present disclosure, such as described in U.S. patentapplication Ser. No. 15/663,600, entitled “Gas-Filled Structures andRelated Compositions, Methods and Systems for Magnetic ResonanceImaging” and filed on Jul. 28, 2017, incorporated herein by reference inits entirety. The MRI can be, for example, T2 type, T2* type, T2 typeweighted, T2* type weighted, QSM type, or Hyper-CEST (Xe). The MRI canbe enhanced by background erasure through ultrasound collapse of theGVPS, can be multiplexed by selected collapse of certain GVPS types,and/or multiplexed by multiparametric unmixing of two or more MRI types(not including Hyper-CEST) where the different GVPS types have differentparametric fingerprints (the ratio of values—susceptibility orrelaxivity—measured by the different MRI types are unique for each GVPStype used). The MRI can be combined with ultrasound imaging to producean enhanced image.

In some embodiments, a hybrid GV gene cluster comprising a combinationof the structural GvpA and GvpC genes from A. floc-aquae with theexpression-enabling GVA secondary genes GvpR-U from B. megateriumresults in the formation of gas vesicles with characteristics favorablefor MRI. For example, in Example 8 an exemplary GVGC construct isreferred to herein as A2C showing robust, acoustically erasable QSMcontrast that was absent from prokaryotic cells that were not induced orprokaryotic cells induced to express a control fluorescent protein.

In particular, in exemplary embodiments where imaging is performed byMRI, the hybrid GV gene cluster can comprise B. megaterium GVA genesGvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU andfurther comprise structural GV proteins genes from Anabaena floc-aquaesuch as GvpA and optionally GvpC (see Example 8). In other embodiments,the hybrid GV gene cluster can comprise B. megaterium GV regulatorygenes GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU andfurther comprise structural GV protein gene GvpA from Bukholderiathailandensis or Psychromonas ingrahamii.

In some embodiments, imaging the target site comprising the prokaryotichost can be performed by applying ultrasound to obtain an ultrasoundimage of the target site.

The term “ultrasound imaging” or “ultrasound scanning” or “sonography”as used herein indicate imaging performed with techniques based on theapplication of ultrasound. Ultrasound refers to sound with frequencieshigher than the audible limits of human beings, typically over 20 kHz.Ultrasound devices typically can range up to the gigahertz range offrequencies, with most medical ultrasound devices operating in the 1 to18 MHz range. The amplitude of the waves relates to the intensity of theultrasound, which in turn relates to the pressure created by theultrasound waves. Applying ultrasound can be accomplished, for example,by sending strong, short electrical pulses to a piezoelectric transducerdirected at the target. Ultrasound can be applied as a continuous wave,or as wave pulses as will be understood by a skilled person.

Accordingly, the wording “ultrasound imaging” as used herein refers inparticular to the use of high frequency sound waves, typically broadbandwaves in the megahertz range, to image structures in the body. The imagecan be up to 3D with ultrasound. In particular, ultrasound imagingtypically involves the use of a small transducer (probe) transmittinghigh-frequency sound waves to a target site and collecting the soundsthat bounce back from the target site to provide the collected sound toa computer using sound waves to create an image of the target site.Ultrasound imaging allows detection of the function of moving structuresin real-time. Ultrasound imaging works on the principle that differentstructures/fluids in the target site will attenuate and return sounddifferently depending on their composition. A contrast agent sometimesused with ultrasound imaging are microbubbles created by an agitatedsaline solution, which works due to the drop in density at the interfacebetween the gas in the bubbles and the surrounding fluid, which createsa strong ultrasound reflection. Ultrasound imaging can be performed withconventional ultrasound techniques and devices displaying 2D images aswell as three-dimensional (3-D) ultrasound that formats the sound wavedata into 3-D images. In addition to 3D ultrasound imaging, ultrasoundimaging also encompasses Doppler ultrasound imaging, which uses theDoppler Effect to measure and visualize movement, such as blood flowrates. Types of Doppler imaging includes continuous wave Doppler, wherea continuous sinusoidal wave is used; pulsed wave Doppler, which usespulsed waves transmitted at a constant repetition frequency, and colorflow imaging, which uses the phase shift between pulses to determinevelocity information which is given a false color (such as red=flowtowards viewer and blue=flow away from viewer) superimposed on agrey-scale anatomical image. Ultrasound imaging can use linear ornon-linear propagation depending on the signal level. Harmonic andharmonic transient ultrasound response imaging can be used for increasedaxial resolution, as harmonic waves are generated from non-lineardistortions of the acoustic signal as the ultrasound waves insonatetissues in the body. Other ultrasound techniques and devices suitable toimage a target site using ultrasound would be understood by a skilledperson.

Types of ultrasound imaging of biological target sites include abdominalultrasound, vascular ultrasound, obstetrical ultrasound,hysterosonography, pelvic ultrasound, renal ultrasound, thyroidultrasound, testicular ultrasound, and pediatric ultrasound as well asadditional ultrasound imaging as would be understood by a skilledperson.

Applying ultrasound refers to sending ultrasound-range acoustic energyto a target. The sound energy produced by the piezoelectric transducercan be focused by beamforming, through transducer shape, lensing, or useof control pulses. The soundwave formed is transmitted to the body, thenpartially reflected or scattered by structures within a body; largerstructures typically reflecting, and smaller structures typicallyscattering. The return sound energy reflected/scattered to thetransducer vibrates the transducer and turns the return sound energyinto electrical signals to be analyzed for imaging. The frequency andpressure of the input sound energy can be controlled and are selectedbased on the needs of the particular imaging task and, in some methodsdescribed herein, collapsing GVs. To create images, particularly 2D and3D imaging, scanning techniques can be used where the ultrasound energyis applied in lines or slices which are composited into an image.

In some embodiments, the ultrasound imaging herein described cancomprising collapsing a GV type expressed in the genetically engineeredbacteria by applying collapsing ultrasound to the target site and/orimaging a GV type in the contrast agent by applying imaging ultrasoundto the target site.

In some embodiments, imaging the target site can be performed byscanning an ultrasound image of the target site in a subject. In somecases, imaging the target site includes transmitting an imagingultrasound signal from an ultrasound transmitter to the target site, andreceiving a set of ultrasound data at a receiver. The visible image isformed by ultrasound signals backscattered from the target site. Theultrasound data can be analyzed using a processor, such as a processorconfigured to analyze the ultrasound data and produce an ultrasoundimage from the ultrasound data. In certain embodiments, the ultrasounddata detected by the receiver includes an ultrasound signal, anultrasound signal reflected by the target site of the subject.

In certain embodiments, the method includes applying a set of imagingpulses from an ultrasound transmitter to the target site, and receivingultrasound signal at a receiver. In certain instances, the ultrasoundsignal detected by the receiver includes an ultrasound echo signal.Additional information of ultrasound systems and methods can be found inrelated publications as will be understood by a person skilled in theart.

Methods for performing ultrasound imaging are known in the art and canbe employed in methods of the current disclosure. In certain aspects, anultrasound transducer, which comprises piezoelectric elements, transmitsan ultrasound imaging signal (or pulse) in the direction of the targetsite. Variations in the acoustic impedance (or echogenicity) along thepath of the ultrasound imaging signal causes backscatter (or echo) ofthe imaging signal, which is received by the piezoelectric elements. Thereceived echo signal is digitized into ultrasound data and displayed asan ultrasound image. Conventional ultrasound imaging systems comprise anarray of ultrasonic transducer elements that are used to transmit anultrasound beam, or a composite of ultrasonic imaging signals that forma scan line. The ultrasound beam is focused onto a target site byadjusting the relative phase and amplitudes of the imaging signals. Theimaging signals are reflected back from the target site and received atthe transducer elements. The voltages produced at the receivingtransducer elements are summed so that the net signal is indicative ofthe ultrasound energy reflected from a single focal point in thesubject. An ultrasound image is then composed of multiple image scanlines.

In some embodiments, imaging the target site is performed by applying ortransmitting an imaging ultrasound signal from an ultrasound transmitterto the target site and receiving a set of ultrasound data at a receiver.The ultrasound data can be obtained using a standard ultrasound device,or can be obtained using an ultrasound device configured to specificallydetect the contrast agent used. Obtaining the ultrasound data caninclude detecting the ultrasound signal with an ultrasound detector. Insome embodiments, the imaging step further comprises analyzing the setof ultrasound data to produce an ultrasound image.

In certain embodiments, the ultrasound signal has a transmit frequencyof at least 1 MHz, 5 MHz, 10 MHz, 20 MHz, 30 MHz, 40 MHz or 50 MHz. Forexample, an ultrasound data is obtained by applying to the target sitean ultrasound signal at a transmit frequency from 4 to 11 MHz, or at atransmit frequency from 14 to 22 MHz.

In the embodiments herein described, the collapsing ultrasound andimaging ultrasound are selected to have a collapsing pressure and animaging pressure amplitude based on the acoustic collapse pressureprofile of the GV type expressed in the prokaryotic cells comprising aGVR genetic circuit. The collapsing ultrasound is typically provided ata high ultrasound pressure amplitude in order to collapse the GVs, whilethe imaging ultrasound is typically provided at a low ultrasoundpressure amplitude to avoid collapsing of the GVs.

In some embodiments herein described, when collapsing ultrasound is usedin combination with ultrasound imaging, acoustically collapsing a GVtype expressed in a prokaryotic cell can remotely in situ erase the GVtype to enable a background-free ultrasound imaging. The background-freeultrasound imaging removes background noise posed by background contrastfrom endogenous sources [35, 36] by subtracting the background imagefrom the GV contrasted image, thus providing higher structure contrastin a final image and increased sensitivity of visualization of the GVs.

In some embodiments, a method is described to provide imaging of one ormore biochemical events in a prokaryotic cell comprised in an imagingtarget site, the method comprising:

introducing into the prokaryotic cell a hybrid gas vesicle reporter genecluster (GVGC) encoding a gas vesicle (GV) type, the GVGC introduced toprovide a reportable genetic molecular components of a GVR geneticcircuit, in which molecular components are connected one to another inaccordance with a circuit design by activating, inhibiting, binding orconverting reactions to form a fully connected network of interactingcomponents, wherein in the reportable genetic molecular component thegas vesicle (GV) type when the genetic circuit operates according to thecircuit design in response to the one or more biochemical events,

wherein the GV type has a selectable acoustic collapse pressure valuederived from an acoustic collapse pressure profile of the GV type and ahydrostatic collapse pressure profile, and a midpoint of the acousticcollapse pressure profile higher than a midpoint of the hydrostaticcollapse pressure profile, and

collapsing the GV type by applying collapsing ultrasound to a targetsite comprising the prokaryotic cell, the collapsing ultrasound appliedat a collapsing ultrasound pressure greater than the selectable acousticcollapse pressure value.

The method further comprises imaging the target site comprising theprokaryotic cell by MRI and/or by applying imaging ultrasound to thetarget site.

In embodiments where imaging is performed by ultrasound, the imagingultrasound is typically a low-pressure ultrasound, applied at an imagingultrasound pressure lower than a selectable acoustic collapse pressurevalue. The selectable acoustic collapse pressure value is selected fromthe acoustic collapsing profile of the GV type expressed by theprokaryotic cells in the target site.

In some of those embodiments, the imaging ultrasound transmit pulses areselected to have an imaging ultrasound pressure equal to or lower thanan initial collapse pressure in the acoustic collapse profile of the GVtype expressed by the prokaryotic cells in the target site.

In some of those embodiments, the imaging ultrasound transmit pulses areselected to an imaging ultrasound pressure equal to or lower than amidpoint collapse pressure in the acoustic collapse profile of the GVtype expressed by the prokaryotic cells in the target site.

In some of those embodiments, the imaging ultrasound transmit pulses areselected to have an imaging ultrasound pressure equal to or lower than acomplete collapse pressure in the acoustic collapse profile of the GVtype expressed by the prokaryotic cells in the target site.

In some of those embodiments, the target site can be treated withcollapsing ultrasound to collapse the GV type expressed by theprokaryotic cells in the target site prior to or after the imaging.

Additional methods to perform imaging of one or more GV types throughultrasound detection alone which are applicable in the presentdisclosure, such as ultrasound imaging with GVs as contrast agents,background erasure of ultrasound imaging by GV ultrasound collapse, andmultiplexing ultrasound imaging of multiple GV types by selectiveultrasound collapse, all of which are described in U.S. patentapplication Ser. No. 15/613,104 filed on Jun. 2, 2017 and incorporatedherein by reference in its entirety.

Accordingly, in the present disclosure, a GV type or types herein can beused as a contrast agent in MRI and/or ultrasound imaging which allowsnon-toxic, highly sensitive and robust contrast at sub-nanomolarconcentrations, with the optional ability of background erasure and/ormultiplexing as would be understood by a skilled person upon reading ofthe present disclosure.

In some embodiments, a method is described to image a biochemical eventin a prokaryotic host comprised in an imaging target site, the methodcomprising:

introducing into the bacterial host a hybrid gas vesicle gene cluster(GVGC) herein described configured for expression in the prokaryotichost, the hybrid gas vesicle gene cluster (GVGC) encoding a gas vesicle(GV) type, wherein the GV type is a reportable molecular component of agas vesicle reporting (GVR) genetic circuit, in which molecularcomponents are connected one to another in accordance with a circuitdesign by activating, inhibiting, binding or converting reactions toform a fully connected network of interacting components, wherein in theGVR genetic circuit an expression of the GV type or an intracellularspatial translocation of the GV type occurs when the GVR genetic circuitoperates according to the circuit design in response to the biochemicalevent; and

imaging the target site comprising the prokaryotic host by applying amagnetic field and/or ultrasound to obtain an MRI and/or an ultrasoundimage of the target site.

In some embodiments, a GVGC can be introduced into a bacterial cell toprovide the bacterial cell with an expressed reportable molecularcomponent comprising a GV type, rendering the cell detectable usingcontrast-enhanced imaging techniques described herein. In theseembodiments, expression of the GV type in the genetically engineeredbacterial cell allows labeling of a target prokaryotic host through aGVR genetic circuit operating according to the circuit design inresponse to a trigger molecular component within the target prokaryotichost.

Accordingly, in some embodiments, a method is described to provide amagnetic resonance and/or ultrasound imaging of a target prokaryoticcell comprised in an imaging target site, to label a target prokaryotichost, the method comprising:

introducing into the target prokaryotic host a hybrid gas vesicle genecluster (GVGC) herein described configured for expression in thebacteria host, the hybrid gas vesicle gene cluster (GVGC) encoding a gasvesicle (GV) type, wherein the GV type is a reportable molecularcomponent of a gas vesicle reporting (GVR) genetic circuit, in whichmolecular components are connected one to another in accordance with acircuit design by activating, inhibiting, binding or convertingreactions to form a fully connected network of interacting components,wherein in the GVR genetic circuit an expression of the GV type or anintracellular spatial translocation of the GV type occurs when the GVRgenetic circuit operates according to the circuit design in response toa trigger molecular component within the target prokaryotic host;

the introducing performed under conditions resulting in presence of thetrigger molecular component in the prokaryotic host.

In some embodiments, the method can further comprise imaging the targetsite comprising the prokaryotic host, by applying a magnetic fieldand/or ultrasound to obtain an MRI and/or an ultrasound image of thetarget site. In particular, in some embodiments the imaging can beperformed at same or different time intervals and/or in different targetsites (e.g. target sites contiguous in space) to detect a spatiallocation of a labeled prokaryotic host.

In embodiments methods to label a target prokaryotic host, expression ofthe GV type occurs when the GVGC genetic circuit operates according tothe circuit design in response to a trigger molecular component in thetarget bacterial cell. The trigger molecular components in the targetbacterial cell that allow activation of the promoter comprised in theGVGC genetic molecular component that regulates initiation of expressionof the GV type comprises transcription factors and other molecularcomponents native to the prokaryotic cell. In some embodiments,heterologous molecular components can also be introduced into the cellto allow activation of expression of the GV type. In some embodiments,the GVGC genetic molecular component comprises a constitutively activepromoter comprising binding sites for transcription factors native tothe target prokaryotic cell. In other embodiments, the GVGC geneticmolecular component comprises an inducible promoter. Exemplaryconstitutive and inducible bacterial promoters suitable for regulatingexpression of GVs in a bacterial cell comprise T7, T7lac, Sp6, araBAD,trp, lac, Ptac, pL, and others identifiable by those skilled in the artand described herein.

In some embodiments, the expression of the GV type in the prokaryotichost when the GVR genetic circuit operates according to the circuitdesign in response to the biochemical event and/or the trigger molecularcomponent in the prokaryotic host allows the selective labeling of aselected prokaryotic host cell type.

In these embodiments, the GVGC genetic circuit is designed tospecifically regulate expression of the GV type in a specific type ofprokaryotic cell. In some embodiments, the GVR genetic circuit isdesigned to comprise genetic molecular components having promoters thatare selectively activated by prokaryote species-specific transcriptionfactors. For example, in embodiments wherein the labeling of E. colicells is specifically required, the GVR genetic circuit can be designedto comprise promoters having E. coli-specific promoters that will not beactivated in prokaryotic cells of another species. Species-specificpromoters are identifiable by those skilled in the art. In otherembodiments, a selected prokaryotic species can be specificallyengineered to control regulation of a GVGC genetic circuit byintroducing additional heterologous genetic molecular components intothe selected prokaryotic host cell, as described herein. Thus, in theseembodiments, specific labeling of the selected prokaryotic speciesallows selective imaging of the specific prokaryotic host usingcontrast-enhanced imaging techniques described herein.

In some embodiments, the expression of the GV type in the prokaryotichost when the GVR genetic circuit operates according to the circuitdesign in response to the biochemical event and/or the trigger molecularcomponent in the prokaryotic host allows the detection of movement andtracking of the location of the prokaryotic host cell within an imagingtarget site. In these embodiments, the tracking of movement of a labeledprokaryotic cell comprises performing serial imaging of one or moreimaging target sites comprising the labeled prokaryotic cell, whereinthe changes in location of the labeled prokaryotic cell in the seriallycollected images of the labeled prokaryotic cell in the one or moreimaging target sites relative to the structures comprised in theenvironment surrounding the labeled prokaryotic cell in the one or moreimaging target sites indicate the movement of the labeled prokaryoticcell within the imaging target site. Exemplary tracking of movementcomprises movement of labeled bacteria within the colon (e.g., Nissle)of a mammalian host or tracking the location of a labeled prokaryoticcell in relation to a tumor in a mammalian host or within blood (e.g.Salmonella) or lymph. In some exemplary embodiments described herein,imaging of engineered bacterial cells expressing GV types in vivo allowsimaging of the location of engineered bacteria in target sites such asgastrointestinal tract and tumors (see Examples 6-7). In severalembodiments, acoustic erasing of one or more GV types can be used toconfirm the specificity of the imaging of the labeled prokaryotic cellwithin the one or more imaging target sites.

The ability of GVs to act as a contrast agent for both ultrasound andmagnetic imaging allows them to act as an acoustomagnetic reporter, thuscreating possibilities for multimodal imaging. In some embodimentsherein described, when collapsing ultrasound is used in combination withMRI imaging, acoustically collapsing a GV type expressed in aprokaryotic cell can remotely in situ erase the GV type to enable abackground-free magnetic resonance imaging of a target site. Thebackground-free magnetic resonance imaging removes background noiseposed by background contrast from endogenous sources [35, 36] byallowing GV types to be identified specifically based on their acousticresponses.

In some embodiments, the ability of GVs to act as a contrast agent witha distinguishable parametric fingerprint for susceptibility-based MRIand/or to allow MRI imaging in combination with ultrasound collapsingallows for the detection of multiple GV types intracellularly as will beunderstood. A parametric fingerprint is a GV types relative (ratio) ofresponse strength for a given concentration for two or more of theparameters: susceptibility, r2 relaxivity, and r2* relaxivity.

Accordingly, in various embodiments herein described imaging of abiochemical event and/or labeling of a prokaryotic cell can be performedby multiplex imaging as will be understood by a skilled person uponreading of the present disclosure.

The term “multiplex” refers to the presence of two or more GV types,each of which exhibits an acoustic collapse pressure profilesubstantially distinct from one another and/or MRI parametricfingerprint substantially distinct one from the other.

In particular, in some embodiments, methods for acoustomagneticmultiplexed imaging of a target site herein described comprise a MRIimaging method to be used in combination with ultrasound collapsing on atarget site contrasted with a contrast agent comprising at least a firstGV type and a second GV type are described in U.S. patent applicationSer. No. 15/613,104 and US patent application Ser. No. 15/663,600,entitled “Gas-Filled Structures and Related Compositions, Methods andSystems for Magnetic Resonance Imaging” and filed on Jul. 28, 2017, bothincorporated herein by reference in their entirety.

In some embodiments of the multiplexed imaging methods herein described,a magnetic resonance imaging of two or more biochemical events in oneprokaryotic cell type comprised in an imaging target site is described.In some of these embodiments, the one prokaryotic cell type comprises aGVR genetic circuit, wherein in the GVR genetic circuit at least twomolecular components are a first GV type and a second GV type when theGVR genetic circuit operates according to the circuit design in responseto the two or more biochemical events. In other embodiments, the oneprokaryotic cell type comprises at least a first GVR genetic circuit anda second GVR genetic circuit, wherein in the first GVR genetic circuitat least one molecular component comprises a first GV type and whereinin the second GVGC genetic circuit at least one molecular componentcomprises a first GV type, when the first and second GVR geneticcircuits operate according to the circuit designs in response to the twoor more biochemical events.

In some embodiments wherein two different GV types are expressed in oneprokaryotic cell, a GVR genetic circuit can be introduced into theprokaryotic cell that is configured to provide alternating expression ofthe two different GV types in the cell, wherein the alternation ofexpression between a first GV gene cluster and a second GV gene clusteris in response to one or more biochemical events in the prokaryoticcell. For example, in some embodiments, a first GV gene cluster can beoperatively linked to a promoter and expression of the first GV typeoccurs in response to a biochemical event that directly or indirectlyactivates the promoter operatively linked to the first GV type; inaddition, in response to a second biochemical event, the expression ofthe first GV type is inactivated and the expression of the second GVtype is activated. An exemplary configuration of a construct comprisingtwo GV gene clusters encoding two different GV types configured foralternating expression of the two GV types is shown in Example 11,wherein a promoter is placed in between a first GV gene cluster and asecond gene cluster, the two GV gene clusters oriented in oppositedirections, and the promoter is flanked by recombination sites thatmediate the reversal of orientation of the promoter to alternate fromoperative connection with the first GV gene cluster and the second GVgene cluster. Thus, for example, upon the occurrence of the secondbiochemical event, the recombinase is expressed and mediatesre-orientation of the promoter to be in operative connection with thesecond GV gene cluster.

In other embodiments of the multiplexed imaging methods hereindescribed, a magnetic resonance imaging of one or more biochemicalevents in each of two or more prokaryotic cell types comprised in animaging target site is described. In some of these embodiments, the twoor more prokaryotic cell types each comprises a GVR genetic circuit,wherein in a first GVR genetic circuit comprised in a first prokaryoticcell type at least one molecular component is a first GV type andwherein in a second GVR genetic circuit comprised in a secondprokaryotic cell type at least one molecular component is a second GVtype when the first and second GVR genetic circuits operate according tothe circuit designs in response to the two or more biochemical events.

Thus, in several embodiments, the multiplexed imaging methods describedherein can be used to independently detect expression of two or moredifferent types of GVs having distinct acoustic collapse profiles. Inexemplary embodiments described herein (see Example 4), multiplexedimaging allows the imaging of more than one population of GV-expressingbacterial cells in a target site. Further, in these embodiments, aspectral unmixing method as described herein and U.S. application Ser.No. 15/613,104, filed on Jun. 2, 2017 can be used to detect differentprokaryotic cell type populations expressing distinct GVs in a targetsite. In those embodiments, imaging the target site further comprisesprocessing the produced images using spectral unmixing to obtainspectrally unmixed images. The term “spectral unmixing”, “acousticspectral unmixing” or “pressure spectral unmixing” as used herein refersto a mathematical image processing method for obtaining spectrallyunmixed images by subtracting each sub-population of signals from a sumof signal contributed by each sub-population present in any given pixel(e.g., see Example 4).

In exemplary embodiments described herein (see Example 4), multiplexedimaging allows the imaging of more than one population of GV-expressingbacterial cells in a target site.

GVs from distinct genetic origins can have different shapes and sizesand therefore can be distinguished on the basis of their differentialeffects on T2, T2*, T2-weighted, T2*-weighed, and QSM contrast.Differences in GV morphology result in different nanoscale magneticfield patterns for a given quantity of gas, which can in turn alter theefficiency of aqueous T2 and T2* relaxation. The magnetic susceptibilitycalculated from QSM reports a value primarily dependent on the totalamount of air in the sample, independent of its nanoscale arrangement.Therefore, each type of GV has its own parametric fingerprint.

Accordingly, in some embodiments, a MRI and/or ultrasound multiplexingmethod and system are described to image of two or more biochemicalevents in one or more one prokaryotic cell types comprised in an imagingtarget site, the method comprising:

introducing into the one or more prokaryotic cell types a first hybridgas vesicle reporter gene cluster (GVGC) encoding a first gas vesicle(GV) type to provide a first reportable genetic molecular component ofone or more GVR genetic circuits in which molecular components areconnected one to another in accordance with a circuit design byactivating, inhibiting, binding or converting reactions to form a fullyconnected network of interacting components wherein in the firstreportable genetic molecular component the first gas vesicle (GV) typeis expressed from the first GVGC when the one or more GVR geneticcircuits operate according to the respective circuit design in responseto the first biochemical event;

introducing into the one or more prokaryotic cell types a second hybridgas vesicle reporter gene cluster (GVGC) encoding a second gas vesicle(GV) type, to provide a second reporting genetic molecular component ofthe one or more GVR genetic circuits, wherein in the second reportablemolecular component the second gas vesicle type is expressed from thesecond GVGC when the one or more GVR genetic circuits operate accordingto the respective circuit design in response to the second biochemicalevent, and

imaging the target site comprising the one or more prokaryotic celltypes.

Exemplary methods to perform imaging the target site comprising the oneor more prokaryotic cell types are described in U.S. patent applicationSer. No. 15/613,104 and U.S. patent application Ser. No. 15/663,600,entitled “Gas-Filled Structures and Related Compositions, Methods andSystems for Magnetic Resonance Imaging” and filed on Jul. 28, 2017, bothincorporated by reference herein in their entirety.

In some embodiments, a method and system is described to provide anultrasound imaging of two or more biochemical events in one or more oneprokatyotic cell types comprised in an imaging target site, the methodcomprising:

introducing into the one or more prokaryotic cell types a first hybridgas vesicle reporter gene cluster (GVGC) encoding a first gas vesicle(GV) type to provide a first reportable genetic molecular component ofone or more GVR genetic circuits in which molecular components areconnected one to another in accordance with a circuit design byactivating, inhibiting, binding or converting reactions to form a fullyconnected network of interacting components wherein in the firstreportable genetic molecular component the first gas vesicle (GV) typeis expressed from the first GVGC when the one or more GVR geneticcircuits operate according to the respective circuit design in responseto the first biochemical event;

introducing into the one or more prokaryotic cell types a second hybridgas vesicle reporter gene cluster (GVGC) encoding a second gas vesicle(GV) type, to provide a second reporting genetic molecular component ofthe one or more GVR genetic circuits, wherein in the second reportablemolecular component the second gas vesicle type is expressed from thesecond GVGC when the one or more GVR genetic circuits operate accordingto the respective circuit design in response to the second biochemicalevent,

wherein the first GV type exhibits a first acoustic collapse pressureprofile and a first selectable acoustic collapse pressure value and thesecond GV type exhibits a second acoustic collapse pressure profile anda second selectable acoustic collapse pressure value, and

selectively collapsing the first GV type by applying collapsingultrasound to the target site comprising the one or more prokaryoticcell types, the collapsing ultrasound applied at a first acousticcollapse pressure value equal to or higher than the first selectableacoustic collapse pressure value and lower than the second selectableacoustic collapse pressure value,

imaging the target site containing the second, uncollapsed, GV type byapplying MRI and/or ultrasound imaging to the target site, the imagingultrasound applied at a pressure value lower than the acoustic collapsepressure value of the second GV type.

In some embodiments, a method and system is described to provide an MRIand/or ultrasound imaging of two or more biochemical events in one ormore one prokaryotic cell types comprised in an imaging target site, themethod comprising:

introducing into the one or more prokaryotic cell types a plurality ofhybrid gas vesicle reporter genes (GVGCs) encoding a plurality of gasvesicle (GV) types, the plurality of GVGCs introduced to provide aplurality of reportable genetic molecular components of one or more GVRgenetic circuits, in which molecular components are connected one toanother in accordance with a circuit design by activating, inhibiting,binding or converting reactions to form a fully connected network ofinteracting components, wherein in each reportable genetic molecularcomponent the gas vesicle (GV) type is expressed from the plurality ofGVGCs when the one or more GVR genetic circuits operate according to thecircuit design in response to a biochemical event,

wherein each GV type exhibits i) an acoustic collapse pressure profiledefined as a collapse function from which a collapse amount can bedetermined, and ii) a selectable acoustic collapse pressure value,selectable acoustic collapse pressure values going from a lowestacoustic collapse pressure value to a highest acoustic collapse pressurevalue,

selectively collapsing each GV type to a collapse amount higher than acollapse amount of each remaining GV type by applying collapsingultrasound to the target site comprising the one or more prokaryoticcell types, the collapsing ultrasound applied at a pressure value equalto or higher than the selectable acoustic collapse pressure value of theGV type being collapsed and lower than an acoustic collapse pressurevalue of said each remaining GV type or types.

The method further comprises imaging the target site containing theremaining GV type or types by applying imaging ultrasound to the targetsite, the imaging ultrasound applied at a pressure value lower than alowest acoustic collapse pressure value of said each remaining GV typeor types. The method also comprises repeating the collapsing and theimaging until all GV types are collapsed, thus providing a sequence ofvisible images of the target site, the sequence being indicative ofimage-by-image decreasing remaining GV types.

In some embodiments where imaging is performed by ultrasound, a hybridGV gene cluster comprising a combination of the structural GvpA genefrom A. floc-aquae with the expression-enabling secondary GVA genesGvpR-U from B. megaterium (FIG. 2 Panel A, middle) results in theformation of gas vesicles with characteristics favorable for ultrasound.Indeed, in exemplary embodiments described herein, expression of thishybrid gene cluster results in E. coli with robust ultrasound contrastcompared to green fluorescent protein (GFP) controls (FIG. 2 Panel B,middle). This exemplary GV gene cluster produces gas vesicles withsignificantly larger dimensions compared to the B. megaterium operon andappear to occupy a greater fraction of intracellular volume (FIG. 2Panels C-D, middle). In other exemplary embodiments described herein,the addition of a gene encoding the A. floc-aquae scaffolding proteinGvpC (FIG. 2 Panel A, right) can further enhance the production oflarger gas vesicles (FIG. 2, Panels C-D, right), resulting in wider andmore elongated nanostructures resembling those native to A. floc-aquae[47], and producing stronger ultrasound contrast (FIG. 2 Panel B,right). This exemplary GVGC construct is referred to herein as ARG1 oracoustic reporter gene 1, used herein in exemplary ultrasound imagingmethods (see e.g., Examples 1-7).

In particular, in exemplary embodiments where imaging is performed byultrasound, the hybrid GV gene cluster can comprise B. megaterium GVAgenes GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU andfurther comprise structural GV proteins genes from Anabaena floc-aquaesuch as GvpA and optionally GvpC (see Example 1). In other embodiments,the hybrid GV gene cluster can comprise B. megaterium GVA genes GvpR,GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU and furthercomprise structural GV protein gene GvpA from Bukholderia thailandensisor Psychromonas ingrahamii.

The DNA sequences of the exemplary constructs of hybrid GV clustersencoding acoustic reporter gene 1 (ARG1; SEQ ID NO: 16) and an exemplaryvariant of ARG1 referred to herein as acoustic reporter gene 2 (ARG2;SEQ ID NO:17) are shown in FIG. 14 and FIG. 15.

In some embodiments, a method is described to provide a magneticresonance imaging and an ultrasound imaging of one or more biochemicalevents in a prokaryotic cell comprised in an imaging target site, themethod comprising:

introducing into the prokaryotic cell a hybrid gas vesicle reporter genecluster (GVGC) encoding a gas vesicle (GV) type to provide a reportablegenetic molecular component of a GVR genetic circuit, in which molecularcomponents are connected one to another in accordance with a circuitdesign by activating, inhibiting, binding or converting reactions toform a fully connected network of interacting components, wherein in thereportable genetic molecular component the GV type is expressed by theGVR when the GVR genetic circuit operates according to the circuitdesign in response to the one or more biochemical events, wherein the GVtype has a selectable acoustic collapse pressure value derived from anacoustic collapse pressure profile of the GV type,

imaging the target site comprising the prokaryotic cell, wherein thetarget site comprises water having a water susceptibility and the GVtype has an associated susceptibility and relaxivity property distinctfrom water and a selectable acoustic collapse pressure value derivedfrom an acoustic collapse pressure profile of the GV type, the imagingperformed by applying an external magnetic field to the target site toobtain a MRI image by detecting relaxivity of the water in the targetsite, and

imaging the target site by applying imaging ultrasound to the targetsite to obtain an ultrasound image of the target site, the imagingultrasound applied an imaging ultrasound pressure lower than aselectable acoustic collapse pressure value of the GV type.

In methods herein described, administration of one or more geneticallyengineered bacterial cell types comprising one or more GVR geneticcircuits to a target site to be imaged, can be performed in any waysuitable to deliver the one or more bacterial cells comprising a GVRgenetic circuit to the target site to be imaged.

In some embodiments, in which the target site is the body of anindividual or a part thereof, the one or more genetically engineeredbacterial cell types comprising a GVR genetic circuit can beadministered to the target site locally or systemically.

The wording “local administration” or “topic administration” as usedherein indicates any route of administration by which the one or moregenetically engineered bacterial cell types comprising a GVR geneticcircuit is brought in contact with the body of the individual, so thatthe resulting location of the one or more genetically engineeredbacterial cell types comprising a GVR genetic circuit in the body istopic (limited to a specific tissue, organ or other body part where theimaging is desired). Exemplary local administration routes includeinjection into a particular tissue by a needle, gavage into thegastrointestinal tract, and spreading a solution containing the one ormore genetically engineered bacterial cell types comprising a GVRgenetic circuit on a skin surface.

The wording “systemic administration” as used herein indicates any routeof administration by which the one or more genetically engineeredbacterial cell types comprising a GVR genetic circuit is brought incontact with the body of the individual, so that the resulting locationof the one or more genetically engineered bacterial cell typescomprising a GVR genetic circuit in the body is systemic (not limited toa specific tissue, organ or other body part where the imaging isdesired). Systemic administration includes enteral and parenteraladministration. Enteral administration is a systemic route ofadministration where the substance is given via the digestive tract, andincludes but is not limited to oral administration, administration bygastric feeding tube, administration by duodenal feeding tube,gastrostomy, enteral nutrition, and rectal administration. Parenteraladministration is a systemic route of administration where the substanceis given by route other than the digestive tract and includes but is notlimited to intravenous administration, intra-arterial administration,intramuscular administration, subcutaneous administration, intradermal,administration, intraperitoneal administration, and intravesicalinfusion.

Accordingly, in some embodiments of methods herein described,administering the one or more genetically engineered bacterial celltypes comprising a GVR genetic circuit can be performed topically orsystemically by intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, rectal, vaginal, and oral routes.In particular, the one or more genetically engineered bacterial celltypes comprising a GVR genetic circuit can be administered by infusionor bolus injection, and can optionally be administered together withother biologically active agents. In some embodiments of methods hereindescribed, administering the one or more genetically engineeredbacterial cell types comprising a GVR genetic circuit can be performedby injecting the one or more genetically engineered bacterial cell typescomprising a GVR genetic circuit such as in a body cavity or lumen. Uponexpression of one or more GV types in one or more genetically engineeredbacterial cell types comprised in the target site, the target site canbe contrast imaged.

Accordingly, in some embodiments, a vector comprising one or moregenetic molecular components of a GVR genetic circuit is described,wherein the vector is configured to introduce the one or more geneticmolecular components comprised in a GVR genetic circuit into aprokaryotic cell.

The term “vector” indicates a molecule configured to be used as avehicle to artificially carry foreign genetic material into a cell,where it can be replicated and/or expressed. An expression vector isconfigured to carry and express the material in a cell under appropriateconditions. In some embodiments, a suitable vector can comprise arecombinant plasmid, a recombinant non-viral vector, or a recombinantviral vector. Vectors described herein can comprise suitable promoters,enhancers, post-transcriptional and post-translational elements forexpression in bacteria that are identifiable by those skilled in theart. Vectors suitable for transduction of prokaryotic cells, and inparticular various Gram negative bacterial cell types are known to thoseskilled in the art. In exemplary embodiments herein described, bacterialexpression plasmids contain all the necessary components to allowcloning methods using E. coli, and comprise elements such as a bacterialorigin of replication (ORI) and elements for plasmid maintenance such asantibiotic selection markers and toxin-antitoxin systems, and alsooptionally to allow incorporating the genes into the bacterial genomeusing recombinases such as Lambda Red, and others identifiable by thoseskilled in the art.

Exemplary vectors for bacterial transformation of E. coli and S.typhimurium with genetic molecular components comprising GV geneclusters are described herein in the Examples.

Accordingly, in some embodiments herein described, a geneticallyengineered prokaryotic cell and in particular a genetically engineeredprokaryotic cell comprising one or more GVR genetic circuits isdescribed. In embodiments described herein, any type of Gram negativebacterial cell can be genetically engineered to comprise one or more GVRgenetic circuits herein described.

In particular, as described above, prokaryotic cells that do notnatively express GVs, or bacterial or archaeal cells in which nativeexpression of GVs has been suppressed, for example through geneticknockout techniques known to those skilled in the art, can be used forheterologous expression of the GVR genetic circuits described herein. Inembodiments herein described, exemplary species of bacteria engineeredto express GV types comprised in GVR genetic circuits described hereinare E. coli and S. typhimurium (see Examples). In some embodiments,bacteria that can be engineered to express GV types comprised in GVRgenetic circuits described herein comprise any type of Gram negativebacteria, such as E. coli, Nissle 1997, and Salmonella. Additionalspecies of bacteria that can be used for heterologous expression of GVsdescribed herein are identifiable by those skilled in the art.

In embodiments herein described, a composition is provided. Thecomposition comprises one or more genetic molecular components of a GVRgenetic circuit, vectors, or genetically engineered prokaryotic cellsdescribed herein together with a suitable vehicle.

The term “vehicle” as used herein indicates any of various media actingusually as solvents, carriers, binders or diluents for the one or moregenetic molecular components, vectors, or prokaryotic cells hereindescribed that are comprised in the composition as an active ingredient.In particular, the composition including the one or more geneticmolecular components, vectors, or prokaryotic cells herein described canbe used in one of the methods or systems herein described.

In some embodiments, one or more Gvp genes in the GV gene clustercomprised in a genetic molecular component of a GVR genetic circuit canbe engineered to produce GVs with altered mechanical, acoustic, surfaceand targeting properties in order to achieve enhanced harmonic responsesand multiplexed imaging to be better distinguished from backgroundtissues. In particular in those embodiments, a GvpC gene encoded in aGVGC gene cluster can be engineered to provide a variant GvpC proteinand corresponding variant GV type, as described in U.S. application Ser.No. 15/613,104, filed on Jun. 2, 2017. In exemplary embodimentsdescribed herein, GvpC genes in GVGC gene clusters herein described areengineered to produce genetically encoded GVs in exemplary GVGCconstructs ARG1 and ARG2 having different acoustic collapse pressurevalues (see Example 4).

In some embodiments, a GV can be engineered to tune the related acousticproperties. In particular the engineering can be performed bygenetically engineering a GV having an acoustic collapse pressure aP₀performed to obtain a variant GV with a critical collapse pressure aP₁lower than the aP₀.

In some embodiments of methods to tune the acoustic properties of a GV,the genetically modified GvpC protein can be modified by at least one of

-   -   a) a deletion of the N-terminal region, C-terminal region or        both    -   b) a deletion of 3 or more repeats, in particular starting from        the repeat adjacent to the C-terminus and moving towards the        N-terminus    -   c) a deletion of at least one repeat immediately after the        N-terminus, and    -   d) addition of amino acids such as functional tags    -   e) substitution of a sub-sequence comprising at least nine amino        acids within the GvpC sequence, wherein the substitution refers        to replacement of amino acids in the original GvpC sequence with        any other amino acid sequence, particularly with other amino        acid sequence having sequence similarity lower than 50% with        respect to the sub-sequence within the GvpC sequence,        to obtain a gas vesicle variant with a critical collapse        pressure aP₁ lower than the aP₀.

In some embodiments, a deletion can comprise a deletion of up to all ofthe amino acids of an N-terminal region, one or more repeat regions, ora C-terminal region. In some embodiments, a deletion can comprise adeletion of part of one or more of an N-terminal region, a C-terminalregion, or a repeat region. For example, a deletion can comprise part ofregion 2 and part of repeat region 3, as shown in the Examples(exemplary variant N-rep2to3-C). In some embodiments, a deletion cancomprise a deletion of more than one repeat region.

In some embodiments, a deletion of a gvpC N-terminal region or aC-terminal region can produce a gvpC variant comprised in a GV having alower aP₁ than a deletion of a gvpC repeat region.

In some embodiments, a deletion of a gvpC N-terminal deletion canproduce a gvpC variant comprised in a GV having a lower aP₁ than adeletion of a gvpC C-terminal deletion.

In some embodiments, a deletion of both a gvpC N-terminal region and agvpC C-terminal region can produce a gvpC variant comprised in a GVhaving a lower aP₁ than a deletion of a gvpC N-terminal region or aC-terminal region performed individually.

In some embodiments, a deletion of one or more repeats regions that arein a position further towards the gvpC N-terminus can produce a gvpCvariant comprised in a GV having a lower aP₁ than a deletion of one ormore repeats regions that are in a position further towards the gvpCC-terminus.

In some embodiments herein described, GV variants without GvpC proteinsor with truncated or mutated GvpC proteins exhibit lower collapsepressure compared to the native GVs under both hydrostatic pressure andultrasound (Example 2 of U.S. application Ser. No. 15/613,104 filed onJun. 2, 2017).

For example, the native Ana GVs have a hydrostatic collapse pressureabout 569.85 kPa, while the Ana GV variants free of GvpC proteins andthe Ana GV variants with truncated GvpC proteins have a hydrostaticcollapse pressure about 195.30 kPa and 374.30 kPa, respectively (seeTable 5 of U.S. application Ser. No. 15/613,104 filed on Jun. 2, 2017).The native Ana GVs have an acoustic collapse pressure about 868.81 kPa,while the Ana GV variants free of GvpC proteins and the Ana GV variantswith truncated GvpC proteins have a hydrostatic collapse pressure about571.00 kPa and 657.04 kPa, respectively (see Table 7 of U.S. applicationSer. No. 15/613,104 filed on Jun. 2, 2017).

In some embodiments described herein, a variant GV can be obtained usinga method of directed evolution, and the resulting GV selected using amethod of high-throughput screening. The term “directed evolution” meansa process wherein random mutagenesis is applied to a protein (e.g. gvpAand gvpC), and a selection regime is used to pick out variants that havethe desired qualities, such as selecting for an altered collapsepressure value. In addition, for example, screening of directedevolution gvpA variants can be performed to select GVs having different(e.g. higher) T2/T2* signal for MRI imaging modalities. Accordingly,polynucleotides encoding gvP proteins as described herein can begenetically mutated using recombinant techniques known to those ofordinary skill in the art, including by site-directed mutagenesis, or byrandom mutagenesis such as by exposure to chemical mutagens or toradiation, as known in the art. The advantage of directed evolution isthat it requires no prior structural knowledge of a protein, nor is itnecessary to be able to predict what effect a given mutation will have.In particular, directed evolution can be performed to detect genemutations resulting in increased harmonic signal (reduced mechanicalstiffness of GVs) for ultrasound, and/or for producing a higher T2/T2*signal in MRI imaging.

An example of high-throughput screening of variant GV types expressed ingenetically engineered bacteria is shown in Example 5.

In some embodiments, GV variants without GvpC proteins or with truncatedor mutated GvpC proteins show harmonic signals several-fold higher thanthe native GVs both in vitro and in vivo.

As used herein, the term “harmonic signal” or “harmonic frequency”refers to a frequency in a periodic waveform that is an integer multipleof the frequency of the fundamental signal. In addition, this termencompasses sub-harmonic signals, which are signals with a frequencyequal to an integral submultiple of the frequency of the fundamentalsignal. In ultrasound imaging, the transmitted pulse is typicallycentered around a fundamental frequency, and received signals may beprocessed to isolate signals centered around the fundamental frequencyor one or more harmonic frequencies. In relation to the imaging of GVs,for those natural or modified GVs that are capable of producing harmonicscattering at a particular acoustic pressure, isolating receivedharmonic signals during imaging can improve the fraction of the imagesignal that is due to the GVs rather than background scattering andreflection. Exemplary GV variants showing show harmonic signals severalfold higher than the native GVs comprise GV variants such as ΔGvpC,ΔN&C-term, ΔN-term, ΔC-term, SR1, SR3, ST-GvpC, GvpC-R8, GvpC-RGD,GvpC-LRP, GvpC-mCD7, SR1CERY1, SR3CERY1, ΔN&C-CERY1, WTCERY1, GvpC-ACPP,GvpC-hPRM, N-term-rep1to2-C-term, Nterm-rep1to3-C-term,N-term-rep2to3-C-term and N-term-rep1to4-C-term in U.S. application Ser.No. 15/613,104. FIG. 9 of U.S. application Ser. No. 15/613,104 showsexemplary images and graphed results showing that GV engineering enablesmodulation of harmonic signals in vivo. FIG. 11 of U.S. application Ser.No. 15/613,104 filed on Jun. 2, 2017 shows an exemplary Clustal Omegasequence alignment of exemplary genetically engineered GvpC proteinsdescribed therein.

The term “fundamental signal” or “fundamental wave” refers to theprimary frequency of the transmitted ultrasound pulse.

The term “non-linear signal” refers to a signal that does not obeysuperposition and scaling properties, with regards to the input. Theterm “linear signal” refers to a signal that does obey those properties.One example of non-linearity is the production of harmonic signals inresponse to ultrasound excitation at a certain fundamental frequency.Another example is a non-linear response to acoustic pressure. Oneembodiment of such a non-linearity is the acoustic collapse profile ofGVs, in which there is a non-linear relationship between the appliedpressure and the disappearance of subsequent ultrasound contrast fromthe GVs as they collapse. Another example of a non-linear signal thatdoes not involve the destruction of GVs, is the increase in bothfundamental and harmonic signals with increasing pressure of thetransmitted imaging pulse, wherein certain GVs exhibit a super-linearrelationship between these signals and the pulse pressure. [48]

In some embodiments, the engineered GvpC variants are obtained byfurther linking the native GvpC protein to one or more other proteins,polypeptides, or domains to form a recombinant fusion protein.

Recombinant fusion proteins can be created artificially usingrecombinant DNA technology identifiable by a person skilled in the artof molecular biology. In general, the methods for producing recombinantfusion proteins comprise removing the stop codon from a cDNA or genomicsequence, such as a polynucleotide coding for a GvpC protein or aderivative thereof, then appending the cDNA or genomic sequence of thesecond protein in frame through ligation or overlap extension PCR.Optionally, PCR primers can further encode a linker of one or more aminoacids residues and/or a PCR primer-encoded protease cleavage site placedbetween two proteins, polypeptides, or domains or parts thereof. Theresulting DNA sequence will then be expressed by a prokaryotic cell as asingle protein. A fusion protein can also comprise a linker of one ormore amino acids residues, which can enable the proteins to foldindependently and retain functions of the original separate proteins orpolypeptides or domains or parts thereof. Linkers in protein or peptidefusions can be engineered with protease cleavage sites that can enablethe separation of one or more proteins, polypeptides, domains or partsthereof from the rest of the fusion protein. Other methods forgenetically engineering these recombinant fusion proteins include SiteDirected Mutagenesis (e.g. using Q5 Site-Directed Mutagenesis Kit fromNEB or the QuickChange Lightning Kit from Agilent), Gibson Assembly(e.g. using the NEB Hi-Fi DNA Assembly Kit), Error-prone PCR (e.g.Mutazyme from Agilent) and Golden-Gate assembly (e.g. using the NEBGolden Gate Assembly Mix).

In some embodiments, a gvpC variant can be produced by engineering agvpC protein from any species that encodes a gvpC protein in its genome,or a synthetically designed gvpC protein. In some embodiments, a gvpCprotein is a gvpC protein from Anabaena floc-aquae, Halobacteriumsalinarum, Halobacterium mediterranei, Microchaete diplosiphon or Nostocsp., or homologs thereof, and others identifiable by a skilled person.

In some embodiments herein described one or more GVs (including variantsGVs) can be engineered to include one or more protein tags to providethe GV with additional functionalities. In particular, in someembodiments GVs can be functionalized through genetic modification of aGvp protein (including variants GvpC protein herein described).

In particular, in some embodiments herein described, one or more proteintags can be added through genetic modification of a GvpC protein or avariant thereof in accordance with the present disclosure of a set typeof GV.

The term “tag” as used herein means protein tags comprising peptidesequences introduced onto a recombinant protein. Tags can be removableby chemical agents or by enzymatic means, such as proteolysis orsplicing. Tags can be attached to proteins for various purposes:Affinity tags are appended to proteins so that they can be purified fromtheir crude biological source using an affinity technique. These includechitin binding protein (CBP), and the poly(His) tag. The poly(His) tagis a widely-used protein tag; it binds to metal matrices. Chromatographytags can be used to alter chromatographic properties of the protein toafford different resolution across a particular separation technique.Often, these consist of polyanionic amino acids, such as FLAG-tag.Epitope tags are short peptide sequences which are chosen becausehigh-affinity antibodies can be reliably produced in many differentspecies. These are usually derived from viral genes, which explain theirhigh immunoreactivity. Epitope tags include V5-tag, Myc-tag, HA-tag andNE-tag. These tags are particularly useful for western blotting,immunofluorescence and immunoprecipitation experiments, although theyalso find use in antibody purification. Protein tags can allow specificenzymatic modification (such as biotinylation by biotin ligase) orchemical modification (such as reaction with FlAsH-EDT2 for fluorescenceimaging). Tags can be combined, in order to connect proteins to multipleother components. However, with the addition of each tag comes the riskthat the native function of the protein may be abolished or compromisedby interactions with the tag. Therefore, after purification, tags aresometimes removed by specific proteolysis (e.g. by TEV protease,Thrombin, Factor Xa or Enteropeptidase).

Exemplary tags comprise the following, among others known to personsskilled in the art: Peptide tags, such as: AviTag, a peptide allowingbiotinylation by the enzyme BirA and so the protein can be isolated bystreptavidin (GLNDIFEAQKIEWHE (SEQ ID NO:18)); Calmodulin-tag, a peptidethat can be bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL(SEQ ID NO:19)); polyglutamate tag, a peptide binding efficiently toanion-exchange resin such as Mono-Q (EEEEEE (SEQ ID NO:20)); E-tag, apeptide recognized by an antibody (GAPVPYPDPLEPR (SEQ ID NO:21));FLAG-tag, a peptide recognized by an antibody (DYKDDDDK (SEQ ID NO:22));HA-tag, a peptide from hemagglutinin recognized by an antibody(YPYDVPDYA (SEQ ID NO:23)); His-tag, typically 5-10 histidines that canbe bound by a nickel or cobalt chelate (HHHHHH (SEQ ID NO:24)); Myc-tag,a peptide derived from c-myc recognized by an antibody (EQKLISEEDL (SEQID NO:25)); NE-tag, a novel 18-amino-acid synthetic peptide(TKENPRSNQEESYDDNES (SEQ ID NO:26)) recognized by a monoclonal IgG1antibody, which is useful in a wide spectrum of applications includingWestern blotting, ELISA, flow cytometry, immunocytochemistry,immunoprecipitation, and affinity purification of recombinant proteins;S-tag, a peptide derived from Ribonuclease A (KETAAAKFERQHMDS (SEQ IDNO:27)); SBP-tag, a peptide which binds to streptavidin(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO:28)); Softag 1, formammalian expression (SLAELLNAGLGGS (SEQ ID NO:29)); Softag 3, forprokaryotic expression (TQDPSRVG (SEQ ID NO:30)); Strep-tag, a peptidewhich binds to streptavidin or the modified streptavidin calledstreptactin (Strep-tag II: WSHPQFEK (SEQ ID NO:31)); TC tag, atetracysteine tag that is recognized by FlAsH and ReAsH biarsenicalcompounds (CCPGCC (SEQ ID NO:32)); V5 tag, a peptide recognized by anantibody (GKPIPNPLLGLDST (SEQ ID NO:33)); VSV-tag, a peptide recognizedby an antibody (YTDIEMNRLGK (SEQ ID NO:34)); Xpress tag (DLYDDDDK (SEQID NO:35)); Covalent peptide tags such as: Isopeptag, a peptide whichbinds covalently to pilin-C protein (TDKDMTITFTNKKDAE (SEQ ID NO:36));SpyTag, a peptide which binds covalently to SpyCatcher protein(AHIVMVDAYKPTK (SEQ ID NO:37)); SnoopTag, a peptide which bindscovalently to SnoopCatcher protein (KLGDIEFIKVNK (SEQ ID NO:38)).

In some embodiments, GvpC can be tagged to alter sub-cellularlocalization of intracellularly expressed GVs to specific bacterial cellcompartments, such as the cell membrane (e.g. with myristoylation tagsor palmitoylation tags). An exemplary myristoylation tag from Src Kinasehas the sequence GSSKSKPKDPSQR (SEQ ID NO:39). An exemplarypalmitoylation tag from GAP43 has the sequence MLCCMRRTKQVEKNDEDQKI (SEQID NO:40)

In some embodiments, GvpC proteins can be tagged to allow clustering ofexpressed GV types in genetically engineered bacterial cells. Exemplarytags allowing clustering comprise homodimerizing proteins such as coiledcoils or dimeric fluorescent proteins.

In embodiments described herein, any of the tags of SEQ ID NO:18-40, andother tags described herein and identifiable by those skilled in theart, can comprise one or more amino acid substitutions, insertions, ordeletions that do not alter the function of the tag, and can furthercomprise one or more additional amino acids, up to a maximum tag lengthof 100 amino acids.

In embodiments described herein a GvpC or a variant gvpC can beengineered to attach a tag fused to or inserted into an N-terminalregion, a C-terminal region of a gvpC or a variant gvpC.

In some embodiments, engineering of a GvpC to attach one or more tagscan be performed with or without substantially alter the criticalcollapse pressure of the base GvpC.

For example in some embodiments described herein, a GvpC protein of a GVcan be engineered to attach one or more protein tags or polypeptide tagswhile optionally substantially altering the acoustic collapse pressureof a GV shell comprising the engineered GvpC as compared to a GV shellof a same non-engineered GvpC.

The term “substantially alter” or “substantially decrease” as usedherein means a decrease of more than 10% in acoustic collapse pressure,preferably more than 20% in acoustic collapse pressure.

In some embodiments described herein, an engineered GvpC protein cancomprise one or more protein tags or polypeptide tags. In embodimentsdescribed herein, appending functional residues comprising one or morepolypeptide tags or protein tags to the N-terminus or the C-terminus ofGvpCWT can reduce collapse pressure depending on the length and exactproperties of the amino acid sequence.

In particular, in some embodiments, engineering of a GvpC can be furtherengineered to attach one or more tags up to the C-terminus withoutsubstantially alter the critical collapse pressure as compared todeleting the N- and/or C-terminal regions. In some embodiments, smalltags such as RGD and RDG do not substantially alter the collapsepressure value. In some embodiments, tags comprising longer sequencessuch as LRP (100 residues) decrease acoustic collapse pressure to agreater extent. In some embodiments, tags such as those comprising mCD47cause a substantial decrease in acoustic collapse pressure value. Insome embodiments, appending a His-Tag (e.g. 6 His amino acids) to theN-terminus of the wild-type GvpC sequence does not substantially alterthe acoustic collapse pressure value. In some embodiments, appending agvpC with a Spytag (FIG. 12 of U.S. application Ser. No. 15/613,104filed on Jun. 2, 2017) is an effective method to functionalize GVs withlarge molecules (greater than 100 amino acids in length) such asfluorescent proteins, without substantially altering their collapsepressure value.

In some embodiments, an in-frame insertion or C- or N-terminal fusion ofa protein tag to a gvpC or a variant gvpC can be performed in an N- orC-terminal region of a gvpC or a variant gvpC. An in-frame insertion canbe performed in several steps, by first providing the gvpC- or variantgvpC-coding and the protein tag-coding polynucleotides and performingthe insertion by breaking a bond (typically a phosphodiester bond)between two adjacent nucleotide bases of the first polynucleotide andthen forming new bonds between the gvpC-coding polynucleotide and theprotein tag-coding polynucleotide. For example, the gvpC codingpolynucleotide can be digested with one or more restrictionendonucleases and then the protein tag-coding polynucleotide inserted byligation (e.g., using T7 DNA ligase) into compatible site(s) allowingformation of phosphodiester bonds between the first and secondpolynucleotide bases. Compatible DNA ligation sites can be “sticky”ends, digested with restriction endonuclease producing an overhang (e.g.EcoRI), or can be “blunt ends” with no overhang, as would be understoodby those skilled in the art. A fusion of a polynucleotide encoding a tagcan also be ligated to an N- or C-terminus of a gvpC or a variant gvpCpolynucleotide by ligation (e.g., using T7 DNA ligase) into compatiblesite(s).

In some embodiments, the gvpC- or variant gvpC-coding and the proteintag-coding polynucleotides can be provided within a singlepolynucleotide by design. For example, a tag can be added by insertingthe polynucleotide encoding a protein of interest in a plasmid or vectorthat has the tag ready to fuse at the N-terminus or C-terminus. The tagcan be added using PCR primers encoding the tag; using PCR the tag canbe fused to the N-terminus or C-terminus of the protein-codingpolynucleotide, or can be inserted at an internal location, usinginternal epitope tagging [49], among other methods known to thoseskilled in the art. Other methods such as overlap extension PCR andinfusion HD cloning can be used to insert a tag at a site between theN-terminus and C-terminus of a protein-coding polynucleotide (seeExamples of U.S. application Ser. No. 15/613,104). Optionally, apolynucleotide encoding a ‘linker’ (such as a sequence encoding a shortpolypeptide or protein sequence, e.g., gly-gly-gly or gly-ser-gly can beplaced between the protein of interest and the tag; this can be usefulto prevent the tag from affecting the activity of the protein beingtagged.

The choice of the location where a tag is added to a protein sequencedepends mainly on the structural and functional features of a proteinand the intended downstream methods employing the use of the tag.

In embodiments herein described, the insertion location of a protein tagin a genetically engineered gvpC or variant gvpC is performed atinsertion position selected to have the tag presented on the externalsurface-exposed position of the gvpC or variant gvpC.

Accordingly, in embodiments described herein, GVR genetic circuitscomprising genetically-encoded GV types can be used together withcontrast-enhanced imaging techniques such as ultrasound imaging and/orMRI to detect the location of and/or dynamic biochemical events inprokaryotic cells in an imaging target site, wherein the prokaryoticcells have been genetically engineered to comprise one or more GVRgenetic circuits described herein.

In some exemplary embodiments, this allows monitoring the activity ofvarious natural and engineered signaling circuits in prokaryotic cells,such as bacterial cells. Furthermore, the ability to distinguishdifferent prokaryotic cell type populations, such as bacterialpopulations through acoustic multiplexing of distinct expressed GV typesin some embodiments allows the study of complex bacterial populationdynamics or the monitoring of multiple engineered therapeutic ordiagnostic agents.

In some exemplary embodiments described herein, imaging of engineeredbacterial cells expressing GV types in vivo allows imaging of engineeredbacteria in target sites such as gastrointestinal tract and tumors (seeExamples 6-7). As understood by those skilled in the art, studies of themammalian microbiome are uncovering an increasing number of criticalroles for bacteria in health and disease, ranging from infection andimmunity to nervous system function [50-53]. Additionally, advances insynthetic biology and genome engineering have led to the development ofmicrobial therapeutics and diagnostics for diseases such asgastrointestinal inflammation and cancer [54-63]. The function of bothnatural and engineered microbes depends strongly on their anatomicallocation within the host organism, making it important to monitor theirspatial distribution, viability, proliferation and function inside thebody [64-66]. Such monitoring requires reporter genes that can beproduced by proliferating prokaryotic cells and connected to specificgenetic circuits. However, conventional reporters based on fluorescentand luminescent proteins or radionuclide capture suffer from the poorpenetration of light into tissue or the need to administer radioactivetracers [67-69]. In contrast to these techniques, ultrasound and MRI arewidely available, inexpensive, radiation-free technologies capable ofnoninvasively imaging deep tissues [70]. For example, the spatialresolution of ultrasound is routinely on the order of 100 μm [71, 72]and can approach the single-micron level with recently developedsuper-resolution techniques [73]. With these performance characteristicsand the ability to place signals within an anatomical context,ultrasound is an ideal technique for imaging microbes in vivo.

As described herein, hybrid GVGCs and related GVR genetic circuits,vectors, genetically engineered bacterial cells, compositions, methodsand systems can be used in several embodiments to detect biochemicalevents in prokaryotic cells using ultrasound imaging or MRI. Inparticular embodiments, the hybrid GVGCs and related genetic circuits,vectors, genetically engineered bacterial cells, compositions, methodsand systems described herein enable ultrasound imaging or MRI ofmicrobes inside mammalian hosts (see e.g., Example 6-7).

In exemplary embodiments described herein, hybrid GVGCs are provided byengineering gas vesicle operons for efficient expression in Escherichiacoli and Salmonella typhimurium—two exemplary commensal and pathogenicspecies that are also chasses for the development of microbialtherapeutics. In some embodiments described herein, GV type-expressingprokaryotic cells can be visualized in vivo in settings relevant togastrointestinal (GI) colonization and antitumor therapy. In exemplaryembodiments described herein, expression of GV types can makeprokaryotic cells visible to ultrasound at volumetric concentrationsbelow 0.01%, allowing dynamic imaging of gene expression and otherbiochemical events, and allows the visualization of bacteria in vivo,such as in mouse colons and tumor xenografts as shown in the Examples.

In some embodiments described herein, engineered gas vesicle geneclusters are used as reporter genes for ultrasound, giving this widelyused noninvasive imaging modality the ability to visualize bacteriainside living animals with sub-100 μm resolution. In several embodimentsdescribed herein, hybrid GVGCs allow prokaryotic cells to be detected atconcentrations below 0.005% v/v or 100 prokaryotic cells per ultrasoundvoxel, making this technology relevant to a broad range of studiesinvolving commensal, disease-causing and engineered microbes. Inexemplary embodiments described herein, bacteria are imaged in themurine GI tract and tumor xenografts, demonstrating the ability ofGVGC-expressing prokaryotic cells to be detected within living animalsat relevant concentrations.

In some embodiments, the GVs and variants thereof comprised in GVRgenetic circuits described herein can be used as a contrast agent in themultiplexed contrast-enhanced imaging methods herein described (seee.g., Example 4).

In particular, a combination of different GV types and/or variantsthereof comprised in GVR genetic circuits, can be used as contrastagents, each expressed GV exhibiting a different acoustic collapseprofile with progressively decreased midpoint collapse pressure values.In some cases, the percentage difference between the midpoint collapsepressure values of any given two expressed GVs types is at least twentypercent.

As mentioned above, the hybrid GV gene cluster and related GVR circuit,molecular component, polynucleotidic constructs, vectors, cells andcompositions herein described can be provided as a part of systems toperform any of the above mentioned methods. The systems can be providedin the form of kits of parts. In a kit of parts, one or more the hybridGV gene cluster and related GVR circuit, molecular component,polynucleotidic constructs, vectors, cells and other reagents to performthe methods herein described are comprised in the kit independently. Thehybrid GV gene cluster and related GVR circuit, molecular component,polynucleotidic constructs, vectors, cells can be included in one ormore compositions, and each the hybrid GV gene cluster and related GVRcircuit, molecular component, polynucleotidic construct, vector and cellis in a composition together with a suitable vehicle.

In particular, the components of the kit can be provided, with suitableinstructions and other necessary reagents, in order to perform themethods here disclosed. The kit will normally contain the compositionsin separate containers. Instructions, for example written or audioinstructions, on paper or electronic support such as tapes or CD-ROMs,for carrying out the assay, will usually be included in the kit. The kitcan also contain, depending on the particular method used, otherpackaged reagents and materials (such as. wash buffers and the like).

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to provide magneticresonance imaging with enhanced contrast and molecular sensitivity atsub-nanomolar concentration.

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to allow multiplexedimaging using parametric MRI, and differential acoustic sensitivity andbackground-free MRI when combined with ultrasound.

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to detectclustering-induced changes in MRI contrast also enable the design ofdynamic molecular sensors.

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to allow multiplexing,multimodal detection and/or molecular targeting to help MRI fulfill itspotential as a high-performance modality for molecular imaging.

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to produce non-toxic,robust MRI contrast via differential magnetic susceptibility atnanomolar concentrations.

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to produce dynamic contrastin response to local molecular signals.

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in several embodiments to provide ultrasoundimaging with enhanced harmonic responses, cellular targeting,multiplexing, multimodal detection and/or molecular targeting to helpultrasound fulfill its potential as a high performance modality formolecular imaging.

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems as wellas GvpC variants herein described can be used in several embodiments totrack moving target sites such as bacterial cells within the body of anindividual or other environments.

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used in connection with various applications whereincontrast-enhanced imaging of a target site is desired. For example, thehybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed can be used for visualization of prokaryotic cells inside ahost individual, such as mammalian hosts, facilitating for example thestudy of the mammalian microbiome and the development of diagnostic andtherapeutic prokaryotic cellular agents, among other advantagesidentifiable by a skilled person, in medical applications, as welldiagnostics applications. Additional exemplary applications include usesof the hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindescribed in several fields including basic biology research, appliedbiology, bio-engineering, bio-energy, medical research, medicaldiagnostics, therapeutics, and in additional fields identifiable by askilled person upon reading of the present disclosure.

Further details concerning the hybrid GVGCs, and related geneticcircuits, engineered bacterial cells and methods of the presentdisclosure will become more apparent hereinafter from the followingdetailed disclosure of examples by way of illustration only withreference to an experimental section.

EXAMPLES

The hybrid GVGCs, and related genetic circuits, vectors, geneticallyengineered prokaryotic cells, compositions, methods and systems hereindisclosed are further illustrated in the following examples, which areprovided by way of illustration and are not intended to be limiting.

In particular, the following examples illustrate exemplary methods andprotocols for providing and using hybrid GVGCs, and related geneticcircuits, vectors, genetically engineered prokaryotic cells,compositions, methods and systems. A person skilled in the art willappreciate the applicability and the necessary modifications to adaptthe features described in detail in the present section, to additionalGVGCs, and related genetic circuits, vectors, genetically engineeredprokaryotic cells, compositions, methods and systems according toembodiments of the present disclosure.

The following materials and methods were used:

Chemicals.

All chemicals were purchased from Sigma Aldrich (St. Louis, Mo.) unlessotherwise noted.

Molecular Cloning.

To construct the plasmid for E. coli expression of ARGs, the genecluster encoding B. megaterium gas vesicle (GV) proteins B, R, N, F, G,L, S, K, J, T and U was amplified from pNL29 [19] (gift of Maura Cannon)and cloned into pET28a using Gibson assembly to give pET28-RNFGLSKJTU.The amplicon included an additional 46 bp upstream of the GvpB startcodon and 180 bp downstream of the GvpU stop codon. To generate hybridgene clusters, the genes encoding GvpA and GvpC were amplified from A.floc-aquae and cloned into pET28-RNFGLSKJTU using Gibson assembly. Acontrol gene encoding the green fluorescent protein (GFP) mNeonGreen[74] was similarly constructed in the pET28 vector. For S. typhimuriumexpression, the ARG gene cluster was cloned into pTD103 (gift of JeffHasty). A control plasmid encoding the luxCDABE gene cluster fromPhotorhabdus luminescens on the pTD103 backbone was also a gift of JeffHasty.

Bacterial Expression.

Plasmids expressing ARGs or GFP were transformed into chemicallycompetent E. coli BL21(A1) cells (Thermo Fisher Scientific, Carlsbad,Calif.) and grown in 5 ml starter cultures in LB media with 50 μg/mlkanamycin, 1% glucose for 16 h at 37° C. Large-scale cultures in LBmedia containing 50 μg/ml kanamycin and 0.2% glucose were inoculated1:100 with the starter culture. Cells were grown at 37° C. to OD600=0.5,then induced with 0.5% L-arabinose and 0.4 mM IPTG for 22 h at 30° C.For Salmonella typhimurium expression, the same protocol was followedexcept constructs were electroporated into S. typhimurium ELH1301 (giftof Jeff Hasty) and induction was with 3 nMN-(β-ketocaproyl)-L-homoserine lactone (AHL).

Gas Vesicle Purification and Quantification.

Harvested cells were centrifugated at 350 g in 50 ml conical tubes for 4h with a liquid height <10 cm to prevent collapse of GVs by hydrostaticpressure. For ARG variants that produce a buoyant band of cells, themidnatant was removed and discarded. For ARG variants that do notproduce a buoyant band, the supernatant was discarded. The remainingcells were resuspended in 8 ml Solulyse-Tris #L200500 (Genlantis, SanDiego, Calif.) per 100 ml culture and 250 μl/ml lysozyme, and incubatedfor 1 h at 4° C. with rotation. Subsequently, 10 μl/ml DNAseI was addedto the lysate and incubated for 10 min at 25° C. The lysate wastransferred to 2 ml tubes and centrifugated for 2 h at 400 g at 8° C.The subnatant was removed with a 21.5 G needle, and the supernatantcontaining the GVs was transferred to a clean tube. PBS was added to theGVs in a 3-fold volume excess and centrifugation, removal of subnatantand PBS dilution was repeated 3 times. Purified GVs were quantifiedusing the Micro BCA Protein Assay Kit (Thermo Fisher Scientific,Carlsbad, Calif.). GVs were collapsed with hydrostatic pressure prior toquantification. Bovine serum albumin was used to generate the standardcurve. Absorbance measurements were taken on a Spectramax M5spectrophotometer (Molecular Devices, Sunnyvale, Calif.).

TEM Sample Preparation and Imaging.

Cells expressing ARGs, or purified GVs, were exchanged into water or 10mM HEPES pH8.0 with 150 mM NaCl, respectively, via 3 rounds of buoyancypurification and buffer exchange as described above. Samples weredeposited on Formvar/carbon 200 mesh grids (Ted Pella) that wererendered hydrophilic by glow discharging (Emitek K100X). For purifiedGVs, 2% uranyl acetate was added for staining. The samples were thenimaged on a FEI Tecnai T12 transmission electron microscope equippedwith a Gatan Ultrascan CCD. Images were processed with FIJI [75].

Hydrostatic Collapse Pressure Measurements.

Cells expressing ARGs, or purified GVs, were diluted to OD600=1.0 in PBSand 0.4 ml was loaded into an absorption cell (176.700-QS, Hellma GmbH &Co. KG, Müllheim, Germany). A single valve pressure controller (PCseries, Alicat Scientific, Tucson, Ariz., USA) supplied by a 1.5 MPanitrogen gas source applied hydrostatic pressure in the cell, while amicrospectrometer (STS-VIS, Ocean Optics, Dunedin Fla., USA) measuredthe optical density of the sample at 500 nm. OD500 was measured from 0to 1.2 MPa gauge pressure with a 10 kPa step size and a 7 secondequilibration period at each pressure.

In Vitro Ultrasound Imaging.

Phantoms for imaging were prepared by melting 1% w/v agarose in PBS andcasting wells using a custom 3D-printed template. Cells at 2× the finalconcentration were mixed 1:1 with molten agarose (at 50° C.) andimmediately loaded into the phantom. The concentration of cells wasdetermined prior to loading by measuring their OD600 after exposure to1.2 MPa hydrostatic pressure to eliminate any contribution to lightscattering from GVs. The optical density was then converted intocells/ml using the relationship 1 OD=8×10⁸ cells/ml(http://www.genomics.agilent.com/biocalculators/calcODBacterial.j sp).Ultrasound imaging was performed using a Verasonics Vantage programmableultrasound scanning system and L22-14v 128-element linear arraytransducer (Verasonics, Kirkland, Wash.). The transducer was mounted acomputer-controlled 3D translatable stage (Velmex, Inc., Bloomfield,N.Y.). Image acquisition was performed using conventional B-mode imagingusing a 128 ray lines protocol with a synthetic aperture to form afocused excitation beam. The transmit waveform was set to a frequency of19 MHz, 67% intra-pulse duty cycle, and a one cycle pulse. Samples werepositioned 6 mm from the transducer face, which is the elevation focusof the L22-14v transducer, coupled through a layer of PBS. The transmitbeam was also digitally focused at 6 mm. For imaging, the transmitvoltage was 2 V and the f-number was 3, resulting in a peak positivepressure of 0.4 MPa. Backscattered ultrasound signals were filtered witha 7 MHz bandpass filter centered at 19 Mhz. Signals backscattered fromfour transmit events were summed prior to image processing. Pixel gainwas set to 3 and persistence to 90. For GV collapse, the transmit powerwas 25 V and the f-number was 0.2. This increased the peak positivepressure to >5 MPa. To ensure complete collapse of the volume, thetransmit focus was scanned from 3 mm to 9 mm. Transducer outputpressures were measured using a fiber-optic hydrophone (PrecisionAcoustics, Dorset, UK).

Plate-Based Induction and Optical Imaging.

ARG and GFP constructs were transformed as described above, and thetransformation mix after recovery was plated on two-layer LB-Agarplates. The underlayer contained 50 μg/ml kanamycin, 1.0% L-arabinose,and 0.8 mM IPTG. The overlayer contained 50 μg/ml kanamycin and 0.4%glucose. The overlayer was poured 30 min prior to plating, and eachlayer was 4 mm thick. Plates with transformants were incubated at 30° C.for 20 h and then imaged for white light scattering and greenfluorescence using a Chemidoc MP instrument (Bio-Rad, Hercules, Calif.).

Colony Ultrasound.

ARG and GFP constructs were transformed into BL21(A1) one-shot competentcells (Thermo Fisher Scientific, Carlsbad, Calif.) and plated ontoLB-Agar two-layer inducer plates as described above. Plates were grownat 37° C. for 14 h. The colonies were immobilized by depositing a 4 mmlayer of 0.5% Agarose-PBS gently onto the plate surface. Ultrasoundimaging was performed using a L11-4v128-element linear array transducer(Verasonics, Kirkland, Wash.). The transducer was mounted on acomputer-controlled 3D translatable stage (Velmex, Inc., Bloomfield,N.Y.). Image acquisition was performed using conventional B-mode imagingusing a 128 ray lines protocol with a synthetic aperture to form afocused excitation beam. The transmit waveform was set to a frequency of6.25 MHz, 67% intra-pulse duty cycle, and a four-cycle pulse. Colonieswere positioned 20 mm from the transducer face, which is the elevationfocus of the L11-4v transducer, coupled through a layer of PBS. Thetransmit beam was also digitally focused at 20 mm. For imaging, thetransmit power was 2 V and the f-number was 3, resulting in a peakpositive pressure of 0.61 MPa. For collapse, the voltage was increasedstepwise to 40V to obtain a maximal peak positive pressure of 5.95 MPa.Pixel gain was set to 0.1 and persistence to 20. Cross-sectional imagesof the plate (perpendicular to the plate surface) were acquired atspatial intervals of 250 μm using computer-controlled steps. Thecross-sectional images were processed in MATLAB to form 2D images of theplate surface. First, the cross-sectional images were stacked to producea 3D-volumetric reconstruction of the plate. The signals in a 2 mm sliceof the volume parallel to and centered on the bacterial growth surfaceafter thresholding to eliminate background were then summed, forming a2D projection image of the plate. After ultrasound imaging, imageprocessing, and acoustic phenotype prediction, the colonies were pickedusing 10 μl sterile pipet tips. Each colony was used to inoculate a 5 mlLB+50 μg/ml kanamycin culture. The cultures were mini-prepped andsequenced to determine whether the plasmid contained GFP, ARG1, or ARG2.

In Vivo Ultrasound Imaging.

All in vivo experiments were performed on BALB/c or SCID nude femalemice under a protocol approved by the Institutional Animal Care and UseCommittee of the California Institute of Technology. Ultrasound imagingwas performed. Mice were anesthetized with 1-2% isoflurane, maintainedat 37° C. on a heating pad, depilated over the imaged region, and imagedusing an L22-14v transducer with the pulse sequence described above. Forimaging of E. coli in the gastrointestinal tract (GI), BALB/c mice wereplaced in a supine position, with the ultrasound transducer positionedon the lower abdomen, transverse to the colon. Anatomical landmarksincluding the bladder were used to identify the colon's position. Priorto imaging, buoyancy-enriched E. coli expressing ARG2 or GFP were mixed1:1 with 42° C. 2% agarose-PBS for a final bacterial concentration of10⁹ cells/ml. The agarose was used to help the live bacteria stay inplace within the GI tract. 150 μL of the mixture was introducedrectally. For imaging of S. typhimurium in tumors, hindlimb ovariantumor xenografts were formed in SCID nude mice via subcutaneousinjection of 5×10⁷ OVCAR8 cells with matrigel. After tumors grew todimensions larger than approximately 6 mm (14 weeks), they were injectedwith ARG1-expressing S. typhimurium. (50 μL, 3.2×10⁹ cells/ml). Thetumors were then imaged with ultrasound, with mice in a prone positionwith anesthesia, homeostasis and imaging parameters as described above.

Image Processing.

MATLAB was used to process ultrasound and optical images.Regions-of-interest (ROIs) were defined to capture all the ultrasoundsignal from the phantom well, colon, or tumor region. All in vitrophantom experiments had the same ROI dimensions. For in vivo experimentsROIs were selected consistently to exclude edge effects from the colonwall or skin. Mean pixel intensity was calculated from each ROI, andpressure-sensitive ultrasound intensity was calculated by subtractingthe mean pixel intensity of the collapsed image from the mean pixelintensity of the intact image. For the multiplexed imaging of ARG1 andARG2, acoustic spectral unmixing was performed according to [76] after aspatial averaging filter (kernel size 30×30 pixels or 750×750 μm) wasapplied to reduce noise. Images were pseudo-colored, with maximum andminimum levels adjusted for maximal contrast as indicated inaccompanying color bars.

Reporter Gene Expression—MRI Experiments.

For reporter gene experiments for MRI imaging, a hybrid GV variant washeterologously expressed in E. coli. In this variant, the major Mega GVcoat protein, GvpB, is replaced by two copies of GvpA and one copy ofGvpC from Anabaena floc-aquae and is therefore named A2C[77]. A2C,instead of Mega, was chosen for the reporter gene experiment because itresults in stronger per-cell expression of GVs[77]. The A2C GV genecluster was expressed from a pET28a plasmid (Novagen, Temecula, Calif.)in BL21(A1) cells (Thermo Fisher Scientific, Waltham, Mass.). 400 μMIPTG and 0.5% arabinose were added at OD₆₀₀ between 0.4 and 0.6 toinduce expression. The control green fluorescent protein mNeonGreen[74]was inserted into the same plasmid and followed the identical culturingprotocol. Cell density was measured after collapsing any intracellularGVs to eliminate their contribution to optical scattering. (FIG. 17). Asample of each E. coli specimen at OD₆₀₀˜1.0 was loaded onto aflow-through, 1 cm path-length quartz cuvette (Hellma Analytics,Plainview, N.Y.), which was pressurized by an N₂ cylinder and a digitalpressure controller (Alicat Scientific, Tucson, Ariz.). The pressure wasincremented in 20 kPa steps from 0 to 1.2 MPa and OD₆₀₀ was recordedusing a spectrophotometer (EcoVis, OceanOptics, Winter Park, Fla.).OD₆₀₀ at 1.2 MPa was used to measure cell density. Prior to thepreparation of MRI phantoms, the cells were concentrated bycentrifugation to the indicated density.

In Vitro MRI and Relaxometry.

E. coli cells were embedded in agarose phantoms. 1% agarose stocksolution was prepared in PBS and maintained at 60° C. until use. Thesize of the phantom was ˜18×6×4 cm (length×width×height). Using a custom3D-printed caster, the bottom half was first cast with cylindrical wellsof the size 3×5 mm (diameter×depth) separated by 3 mm. The cylindricalgeometry perpendicular to B₀ was chosen to ensure a homogeneous field inthe sample wells to facilitate susceptibility-based imaging. The gel wasallowed to solidify and exposed to air for 1 h for gas equilibration. E.coli cells in PBS were mixed 1:1 with the melted agarose stock solutionand immediately loaded into the wells. Subsequently, the top half of thephantom was cast so that all the wells were surrounded by agarose. Carewas taken to avoid bubbles. MRI was performed on a 7T horizontal boreBruker-Biospin scanner, using a 7.2 cm diameter volume coil for transmitand receive. T2* relaxivity was measured with 3D Multi Gradient Echo(MGE) experiments with the following parameters: repetition time(TR)=500 ms, 32 echos, echo spacing (TE)=9.0 ms, field of view(FOV)=12×6×0.8 cm³, spatial resolution=0.25×0.25×0.25 mm³ and 1 average.T2 relaxometry was performed by 2D Multi Slice Multi Echo (MSME) spinecho experiments with the following parameters: TR=2500 ms, 16 echos,TE=16.0 ms, FOV=8×6 cm², and spatial resolution=0.25×0.25 mm². Slicethickness=1 mm and 16 averages were used for multiparametricmultiplexing experiments and 0.5 mm and 4 averages for all otherexperiments. T1 relaxometry was performed by 2D Rapid Acquisition withRelaxation Enhancement with Variable TR (RAREVTR) with the followingparameters: Effective TE=9.683 ms, RARE factor=12, FOV=8×6 cm², spatialresolution=0.25×0.25 mm², slice thickness=0.5 mm, 2 average and 8variable TR times including: 126.43, 738.40, 1461.21, 2344.09, 3478.70,5068.54, 7746.55, 20000.00 ms. For data analysis, a circular region ofinterest (ROI) was drawn for each well using Fiji[75]. The averageintensity of the ROI was imported into Matlab for exponential fitting toderive the T2*, T2 and T1 values. Voxel-wise T2* and T2 maps weregenerated by ImageJ plugin, MRI Processor, using Simplex fitting. ForT2* relaxometry, the ROI excluded the rim of the wells.

Quantitative Susceptibility Mapping.

Magnitude and phase images of 3D MGE or 3D fast low angle shot (FLASH)experiments were obtained in ParaVision 5.1 (Bruker), and the imagesfrom a single echo served as the input to the Susceptibility Mapping andPhase artifacts Removal Toolbox (SMART) (MRI Institute for BiomedicalResearch, Detroit, Mich.). This software performed phase unwrappingusing the 3D-SRNCP algorithm[78], background field removal by the SHARPalgorithm[79] and susceptibility map generation using the SWIMalgorithm[80]. The resulting QSM images were analyzed in Fiji[75].Unless specified otherwise, all QSM images were processed from the5^(th) echo (TE=45.0 ms) of a 3D MGE experiment.

In Vitro Acoustic Collapse—MRI Experiments.

For collapsing the intracellular GVs in E. coli, a Verasonics Vantageprogrammable ultrasound scanning system using the L11-4v 128-elementlinear array transducer (Verasonics, Kirkland, Mass.) was used with thefollowing parameters: transmit frequency=6.25 MHz, transmit voltage=15V.

TABLE 4 MRI measurements of E. coli in agarose phantom. All the valueswere zeroed by the PBS sample. Errors represent SEM and N = 6.

 (ppb)

 R2* (sec⁻¹)

 R2 (sec⁻¹) Before US After US Before US After US Before US After USGV + IPTG  1.93 ± 0.31 −2.73 ± 0.26 0.97 ± 0.13 0.45 ± 0.08 0.65 ± 0.030.34 ± 0.04 GFP + IPTG −1.97 ± 0.36 −1.82 ± 0.39 0.32 ± 0.10 0.50 ± 0.090.28 ± 0.04 0.26 ± 0.04 GV −2.75 ± 0.17 −2.00 ± 0.19 0.34 ± 0.10 0.29 ±0.03 0.35 ± 0.05 0.33 ± 0.05 PBS    0 ± 0.14    0 ± 0.16   0 ± 0.09   0± 0.04   0 ± 0.02   0 ± 0.03

Example 1. Genetic Engineering of Acoustic Reporter Genes

Gas vesicles are encoded in their native bacterial or archaeal hosts byoperons of 8-14 genes, which include the primary structural proteinGvpA, the optional external scaffolding protein GvpC, and severalsecondary proteins that function as essential minor constituents orchaperones [11] (FIG. 2 Panel A). It was previously shown that E. colitransformed with a gas vesicle gene cluster from B. megaterium arecapable of producing small bicone-shaped gas vesicles [19]. However, itwas found that expression of this gene cluster in E. coli does notresult in bacteria detectable by ultrasound (FIG. 2 Panel B, left), mostlikely because the small gas vesicles produced from this construct (FIG.2, Panels C-D, left) have poor acoustic properties. At the same time,transforming E. coli with a gas vesicle gene cluster derived from thecyanobacterium Anabaena floc-aquae, whose gas vesicles are highlyechogenic [47, 76], did not yield gas vesicle expression. Given the highsequence homology of GvpA between organisms (FIG. 3), it washypothesized that a combination of the structural GvpA genes from A.flos-aquae with the expression-enabling secondary genes GvpR-U from B.megaterium (FIG. 2 Panel A, middle) would result in the formation of gasvesicles with characteristics favorable for ultrasound imaging. Indeed,expression of this hybrid gene cluster resulted in E. coli with robustultrasound contrast compared to green fluorescent protein (GFP) controls(FIG. 2 Panel B, middle). These cells produced gas vesicles withsignificantly larger dimensions compared to the B. megaterium operon andappeared to occupy a greater fraction of intracellular volume (FIG. 2Panels C-D, middle). The addition of a gene encoding the A. floc-aquaescaffolding protein GvpC (FIG. 2 Panel A, right) further enhanced theproduction of larger gas vesicles (FIG. 2, Panels C-D, right), resultingin wider and more elongated nanostructures resembling those native to A.floc-aquae [47], and producing stronger ultrasound contrast (FIG. 2Panel B, right); this construct is referred to herein as ARG1 oracoustic reporter gene 1.

To confirm that the ultrasound signal from ARG1-expressing cells is dueto the presence of gas vesicles, acoustic pulses were applied withamplitudes above the gas vesicles' critical collapse pressure. Inpurified form, this results in the immediate collapse of these proteinnanostructures and dissolution of their gas contents, eliminatingultrasound contrast [47]. As expected, the application of high-pressurepulses made cells expressing ARG1 invisible to ultrasound (FIG. 2 PanelB). The ability of ARG-based contrast to be erased in situ is usedthroughout this study to confirm the source of acoustic signals andsubtract background.

ARG1 expression resulted in average gas vesicle contents of 9.4±0.4 mg/gE. coli (FIG. 2 Panel E), corresponding to approximately 100 gasvesicles per cell. These nanostructures occupy roughly 10 percent of theintracellular space, which is sufficient to make a subset ofARG1-expressing cells buoyant (FIG. 2 Panel F). Cells expressing theother operons produced a similar quantity of proteins but were notbuoyant, presumably due to the smaller volume-to-surface ratio of theirgas vesicles. These results show that genetic engineering enables thecreation and optimization of ARGs for ultrasound imaging of bacteria.

Example 2. ARGs Enable the Imaging of Dilute Cell Populations andConditional Gene Expression

To enable a broad range of in vivo applications, noninvasive imaging isdesigned be able to detect relatively dilute cellular populations. Forexample, the large intestine, a key target of microbiome research andengineered microbial therapeutics, hosts a bacterial population ofapproximately 10¹⁰ cells/ml [81], representing a volume fraction ofabout 1%. To determine the detection limit of ARG-expressing cells, aconcentration series of E. coli transformed with ARG1 was imaged (FIG. 4Panel A). Cells at concentrations as low as 5×10⁷ cells/ml produceddetectable signal (FIG. 4, Panels A and B). This equates to a roughly0.005% volume fraction, or approximately 100 cells per voxel based oncubic voxel dimensions of 100 μm. This sensitivity should be sufficientfor many in vivo scenarios. Furthermore, bacteria enriched for buoyancyprior to imaging provide 2.4-fold higher signal (FIG. 5), suggestingthat sensitivity could be improved further by optimizing ARG expression.

In addition to observing the spatial distribution of cells, it isdesirable to monitor dynamic cellular signals. Many biological states,signaling pathways and environmental stimuli can be connected to geneexpression, as often done with gene circuits wired to fluorescentindicators [82]. To test whether ARGs could provide a similar readout ofstate-dependent genetic pathways, ARGs were placed under the control ofa promoter regulated by the chemical inducer isopropylβ-D-1-thiogalactopyranoside (IPTG). Ultrasound signals from E. coliexpressing ARG1 in this configuration followed the expecteddose-response curve of IPTG-controlled expression (FIG. 4, Panels C andD), confirming their ability to serve as the output signal forengineered genetic circuits.

Example 3. ARG Expression and Ultrasound Imaging do not Affect CellViability

To determine whether the expression of ARGs has any deleterious effecton host cells, the growth curves of E. coli expressing ARG1 or GFP weremeasured. After induction, cells expressing both constructs continued todivide and reached similar saturation densities (FIG. 6 Panel A). Forboth ARG1 and GFP the final density was somewhat lower than in uninducedcontrols, as expected from the metabolic demand of protein expression[83]. This has not been a major limitation for the use of GFP-basedreporters.

Next, the viability of ARG-expressing cells was assessed afterultrasound imaging and acoustic collapse. TEM images of cells acquiredbefore and after exposure to collapsing acoustic pulses show that gasvesicles can be eliminated without obvious cellular damage (FIG. 6 PanelB). To examine the impact of ultrasound exposure on cell growth, E. coliexpressing ARG1 were cultured as colonies on solid media and acousticcollapse pulses were applied to half the plate. Gas vesicle collapse ininsonated cells was confirmed by a decrease in optical scattering, asseen on dark-field images of the plates (FIG. 6, Panels C-D). Afterincubation for an additional 20 h, no significant difference wasobserved in the diameter of the insonated colonies compared toun-insonated controls, indicating that ultrasound exposure does notaffect cell viability (FIG. 6 Panel E). Strikingly, insonated coloniesre-expressed gas vesicles during this period, as indicated by therestoration of pressure-sensitive light scattering (FIG. 6, Panels E-F).This result suggests that ultrasound could be used for pulse-chasestudies analogous to fluorescence photobleaching recovery assays [84].

Example 4. Engineered Variants of ARGs Enable Multiplexed CellularImaging

It is often informative to simultaneously image more than one populationof cells, for example to monitor the dynamics of competing microbialspecies or the interaction of multiple therapeutic or diagnosticconstructs. Optical multiplexing typically makes use of spectrallydistinct fluorescent proteins, and analogous acoustic multiplexing canbe performed using genetic variants of gas vesicles that collapse atdifferent pressures [76]. Such gas vesicles can be distinguished fromeach other by applying acoustic pulses of gradually increasing amplitudeand monitoring the disappearance of backscattered signal: one subset ofgas vesicles collapses first, followed by another, and so on. A signalprocessing paradigm similar to spectral unmixing then determines thecontribution of each population to the total signal [76]. It washypothesized that if ARGs could be engineered to produce intracellulargas vesicles collapsing at different pressures, this would enablemultiplexed imaging of distinct cellular populations.

To explore this possibility, a new version of the ARG gene clustercontaining a modified version of A. floc-aquae GvpC was constructed.Previous work has shown that deletion or truncation of this outerscaffolding protein results in gas vesicles with lower collapsepressures [85, 86], allowing the production of nanostructuresdistinguishable from each other under ultrasound [76]. Following thisapproach, the gene cluster was modified by truncating GvpC to retainonly one of its five repeating alpha-helical domains (FIG. 7 Panel A).E. coli expressing the resulting gene cluster, referred to herein asARG2, showed robust gas vesicle production and ultrasound contrast,similar to ARG1 (FIG. 7, Panels B to D, FIG. 8). Consistent with thedesign, gas vesicles purified from ARG2-expressing E. coli had a lowercritical collapse pressure than nanostructures formed by cellsexpressing ARG1 (FIG. 7 Panel E), and cellular ARG2 contrast waserasable at lower acoustic pressures (FIG. 7 Panel F). The two variants'distinct collapse spectra (FIG. 7 Panel G) allowed E. coli expressingARG1 and ARG2 to be imaged in multiplex using pressure spectrum unmixing(FIG. 7, Panels H-I).

Example 5. High-Throughput Screening of Acoustic Reporter Genes

Directed evolution has served as an effective approach to fluorescentprotein engineering to identify variants with new spectral andbiochemical properties [87-89]. This approach typically requires ahigh-throughput screen, which is commonly implemented by plating abacterial library of genetic variants on agar and imaging the resultingcolonies to identify mutants with desired optical properties [88, 89].To determine whether a similar approach could be used with ARGs, amethod was developed to scan bacterial colonies with ultrasound (FIG. 9Panels A-C). In this method, colonies are immobilized on agar plateswith an over-layer of agarose, then scanned with an ultrasoundtransducer translated by a computer-controlled robot. This results in aseries of transverse images that can be reconstructed to form anin-plane image of the plate. To assess the ability of this screeningplatform to discriminate acoustic phenotypes, a mixed plate of E. colitransformed with ARG1, ARG2 or GFP was imaged. Serial acoustic collapseimaging (FIG. 9 Panel B) revealed three distinct colony populations: onelacking ultrasound signal (FIG. 9 Panel D), one collapsing at a lowerpressure, and one collapsing at a higher pressure (FIG. 9 Panel E).Based on these acoustic properties, the ARG1, ARG2 and GFP genotypescould be distinguished from each other with 100% accuracy, as determinedby DNA sequencing (FIG. 9 Panel F). This result confirms that colonyscreening can discriminate acoustic phenotypes with sufficient accuracyto serve as a high-throughput assay for acoustic protein engineering.

Example 6. In Vivo Gastrointestinal Imaging of Engineered Microbes

After establishing the core capabilities of ARGs in vitro, their basicfunctionality in vivo was demonstrated by imaging ARG-expressing cellsin biologically relevant anatomical contexts. One particularly importanttarget for in vivo microbial imaging is the mammalian GI tract, givenrecent findings concerning the impact of the gut microbiome on humanhealth [50, 64-66] and the development of GI-targeted microbialtherapeutics [57, 58, 60, 90, 91]. Due to its location deep inside thebody, the GI tract is difficult to image using optical techniques, andtherefore represents a major opportunity for ultrasound. To establish aproof of concept for the imaging of ARG-expressing E. coli within thegut, cells expressing ARG2 were introduced into the colons of live miceand imaged their abdomens with ultrasound (FIG. 10 Panel A). Backgroundsubtraction after acoustic collapse allowed us to obtain signals fromthe colon that were specific to ARG-labeled cells and absent in GFPcontrols (FIG. 10, Panels B and C, FIG. 11). These signals are overlaidon anatomical images showing the location of the bacteria within thecontext of other internal organs. These results establish the ability ofARGs to make microbial gene expression visible noninvasively in deeptissue.

Example 7. ARG Expression in Salmonella typhimurium and Imaging InsideTumors

Another emerging application of engineered microbes is as antitumortherapies and diagnostics [61, 63, 92]. For example, Salmonellatyphimurium has been engineered to colonize tumors and secrete proteinscausing cancer cell lysis and immune system stimulation [61, 92]. Beingable to image such therapies in the body could facilitate theirdevelopment and clinical translation. To enable this possibility, thegenetic construct encoding ARG1 was adapted for expression in attenuatedS. typhimurium strain ELH1301, which has been used in tumor-homingstudies [61, 92]. Upon induction with N-(β-ketocaproyl)-L-homoserinelactone (AHL), ARG-expressing S. typhimurium cells produced abundantintracellular gas vesicles and were readily observable using ultrasoundcompared to controls expressing the bacterial luciferase operon LuxABCDE(FIG. 12, Panels A-B). The level of ultrasound contrast was similar toARG-expressing E. coli, with mean intensities per pixel of 9.5±0.7 and12.1±2.1, respectively, at a density of 10⁹ cells/ml. Followingpressure-induced collapse, these cells are indistinguishable fromluciferase-expressing controls (FIG. 12 Panel A).

Next, it was tested whether ARG-expressing S. typhimurium could beimaged in vivo in a murine tumor, where these bacteria can proliferateto densities of 10¹⁰ cells per gram tissue [92]. Live ARG-expressingcells were injected into OVCAR8 ovarian adenocarcinoma xenografts innude mice and imaged with ultrasound. Contrast was readily apparent intumors containing engineered S. typhimurium, and disappeared afteracoustic collapse (FIG. 12 Panels C, D). Cells expressing the luciferaseoperon had no discernable ultrasound contrast (FIG. 12 Panel D). Theseresults demonstrate that ARGs can be employed in more than one bacterialspecies and that tumor-homing bacteria are detectable inside tumors atconcentrations relevant to therapeutic and diagnostic applications.

Example 8. Acoustomagnetic Imaging of Gene Expression

Heterologous expression of a GVGC gene cluster comprising a combinationof genes from A. floc-aquae and B. megaterium[77] was placed under thecontrol of a promoter inducible by isopropyl b-D-1-thiogalactopyranoside(IPTG, FIG. 16, Panel A). Overnight induction resulted in GV expressionand robust, acoustically erasable QSM contrast that was absent fromcells that were not induced or cells induced to express a controlfluorescent protein (FIG. 16, Panels B-C). Notably, the E. coliconcentration in the phantom, estimated from OD₆₀₀ to be ˜14 g/L wetcellular weight [93], indicates that the GV-containing cells can bedetected while comprising less than 1.4% of the imaged volume.

Example 9: Amino Acid Sequences of Exemplary GVS and GVA Proteins

Table NF1, NF2 and NF3 show amino acid sequences of exemplary GVS(GvpA/B or GvpC) and GVA proteins from several exemplary prokaryoticspecies. In particular, these exemplary amino acid sequences can be usedas reference amino acid sequences in some embodiments for homology-basedsearches for related GVS and GVA proteins.

TABLE 5 Amino acid sequences of exemplary gvpA and gvpB proteinsSpecies, protein; SEQ ID GenBank accession Amino acid sequence NO.:Anabaena flos-aquae, gvpA; MAVEKTNSSSSLAEVIDRILDKGIVIDAWVRVS 41gi|121860|sp|P10397.3 LVGIELLAIEARIVIASVETYLKYAEAVGLTQSA AVPAB. megaterium, gvpA; MSIQKSTDSSSLAEVIDRILDKGIVIDAFARVSL 42gi|294500059|ref|YP_ VGIEILTIEARVVIASVDTWLRYAEAVGLLTDK 003563759.1VEEEGLPGRTEERGAGLSF B. megaterium, gvpB1;MSIQKSTNSSSLAEVIDRILDKGIVIDAFARVSV 43 gi|294500056|ref|YP_VGIEILTIEARVVIASVDTWLRYAEAVGLLRDD 003563756.1| VEENGLPERSNSSEGQPRFSIFrankia sp, gvpA; MTVSSQSMNRAPKPSSLADVLDVVLDRGIVID 44gi|86739718|ref|YP_480118.1 AYARVALVGIEVLTADARVVIATVDTYLRFAEAVNRLDLAPKEQVPGLPGLMHEVTDGTARQK SKGALEGLKDTAEEAVGSLRGGSSEEHARRDLPAGRSAPGDRRSGREG Haloferax mediterranei, MVQPDSSSLAEVLDRVLDKGVVVDVWARISL45 gvpA; VGIEILTVEARVVAASVDTFLHYAEEIAKIEQAE gi|389847150|ref|YP_LTAGAEAAPTPEA 006349389.1 Halobacterium sp NRC-1,MAQPDSSGLAEVLDRVLDKGVVVDVWARVSL 46 gvpA1;VGIEILTVEARVVAASVDTFLHYAEEIAKIEQAE gi|16120003|ref|NP_395591.1LTAGAEAAPEA Halobacterium sp NRC-1, MAQPDSSSLAEVLDRVLDKGVVVDVWARISL 47gvpA2; VGIEILTVEARVVAASVDTFLHYAEEIAKIEQAE gi|16120172|ref|NP_395760.1LTAGAEAPEPAPEA Halorubrum vacuolatum, MAQPDSSSLAEVLDRVLDKGVVVDVYARLSL 48gvpA; VGIEILTVEARVVAASVDTFLHYAEEIAKIEQAE gi|22095734|sp|O33397.1LTAGAEAAPTPEA Microcystis aeruginosa MAVEKTNSSSSLAEVIDRILDKGIVIDAWARVS49 NIES-843, gvpA; LVGIELLAIEARVVIASVETYLKYAEAVGLTQSgi|166366499|ref|YP_ AAVPA 001658772.1 Methanosarcina barkeri,MVSQSPDSSSLAEVLDRILDKGIVVDTWARVSL 50 gvpA;VGIEILAIEARVVVASVDTFLHYAEEITKIEIAAR gi|73667875|ref|YP_303890.1 EEKPAIAASerratia sp. ATCC 39006, MAKVQKSTDSSSLAEVVDRILDKGIVIDAWVK 51 gvpA1;VSLVGIELLSIEARVVIASVETYLKYAEAIGLTA gi|555225836|gb|ESN63289.1 SAATPASerratia sp. ATCC 39006, MPVNKQYQDEQQQVSLCEALDRVLNKGVVIV 52 gvpA2;ADITISVANIDLIYLSLQALVSSVEAKNRLPGRE gi|555225844|gb|ESN63297.1Serratia sp. ATCC 39006, MSGNKKLTHSTDSTTVADLLERLLDKGVVISG 53 gvpA3;DIRIRLVEVELLTLEIRLLICSVDKAVEMGLDW gi|555225847|gb|ESN63300.1WSGNPAFDSRARVSSSAPAPELEERLQRLEARL EAAPSVIEETHL Streptomyces coelicolor,MITYDDEVVCAPRAGTLYDVLELILDRGMVID 54 gvpA1;VFVRVSLVGIEILKVDARIVVASVDTYLRFAEA gi|21219180|ref|NP_624959.1CNRLDLEHDVRSKTVPEMFGSPMAKTVGRAG ARRTARSLTDKVRDVLTPEHEHEEEPEEAEDRPRAGAERGRSTQRPRSRPAARPRDEDDRPRSRPR RRTEEEDR Streptomyces coelicolor,MTVVPAQQTGGGGSSGLYDVLELVLDRGLVID 55 gvpA2;AFVRVSLVGIEILKIDVRVVVASVDTYLRFAEA gi|21224803|ref|NP_630582.1CNRLDLEAGPRKDPGLPDLVGEMTESGARGKS KGALSGAAETISDAFKQARDDGGSERETSSRPRARKAAPSRRKEEQE Bukholderia, gvpA1 MAKVQKSTDSSSLAEVVDRILDKGIVIDVWAK 56VSLVGIELLSIEARVVIASVETYLKYAEAIGLTA TAAAPTA Bukholderia, gvpA2MADLLERVLDKGVVITGDIRINLVDVELLTIRIR 57 LLVCSVDKAKELGIDWWNADTFFLGPDRGQSALPGRASAVDVAAGSAVHADAAHR Psychromonas gvpA1MANVQKSTDSSGLAEVVDRILEKGIVIDAFVKV 58 SLVGIELLSIEARVVIASVETYLKYAEAIGLTASAATPA Psychromonas gvpA2 MANVQKTTDSSGLAEVIDRILDKGIVIDAFVKV 59SLVGIELLSIEARVVIASVETYLKYAEAIGLIAS AATPA Psychromonas gvpA3MATGKPQSMTHSVKSTTVADLLERILDKGIVV 60 TGDIKIKLVDVELLTVELRLVICSVDKAVEMGMDWWNNNPAFAPQAPAQEGELSSIEKRLEKIE KALVK Psychromonas gvpA4MPMANVSINPELTAQECEKISLCDALDRIINKG 61 VVIHGEITISVANVDLISLGVRLILSNVETREQSNTPKEEV

TABLE 6 Protein sequences of gvpC from exemplary species: UniProt SEQSpecies ID No. Amino acid Sequence ID NO: Anabaena flos- P09413MISLMAKIRQEHQSIAEKVAELSLETREFLSVTTAK 62 aquaeRQEQAEKQAQELQAFYKDLQETSQQFLSETAQARI AQAEKQAQELLAFHKELQETSQQFLSATAQARIAQAEKQAQELLAFYQEVRETSQQFLSATAQARIAQAE KQAQELLAFHKELQETSQQFLSATADARTAQAKEQKESLLKFRQDLFVSIFG Halobacterium P24574MSVTDKRDEMSTARDKFAESQQEFESYADEFAADI 63 salinarumTAKQDDVSDLVDAITDFQAEMTNTTDAFHTYGDE FAAEVDHLRADIDAQRDVIREMQDAFEAYADIFATDIADKQDIGNLLAAIEALRTEMNSTHGAFEAYADD FAADVAALRDISDLVAAIDDFQEEFIAVQDAFDNYAGDFDAEIDQLHAAIADQHDSFDATADAFAEYRD EFYRIEVEALLEAINDFQQDIGDFRAEFETTEDAFVAFARDFYGHEITAEEGAAEAEAEPVEADADVEAE AEVSPDEAGGESAGTEEEETEPAEVETAAPEVEGSPADTADEAEDTEAEEETEEEAPEDMVQCRVCGEYY QAITEPHLQTHDMTIQEYRDEYGEDVPLRPDDKTHalobacterium Q02228 MSVKDKREKMTATREEFAEVQQAFAAYADEFAA 64 mediterraneiDVDDKRDVSELVDGIDTLRTEMNSTNDAFRAYSE EFAADVEHFHTSVADRRDAFDAYADIFATDVAEMQDVSDLLAAIDDLRAEMDETHEAFDAYADAFVTD VATLRDVSDLLTAISELQSEFVSVQGEFNGYASEFGADIDQFHAVVAEKRDGHKDVADAFLQYREEFHGV EVQSLLDNIAAFQREMGDYRKAFETTEEAFASFARDFYGQGAAPMATPLNNAAETAVTGTETEVDIPPIE DSVEPDGEDEDSKADDVEAEAEVETVEMEFGAEMDTEADEDVQSESVREDDQFLDDETPEDMVQCLVC GEYYQAITEPHLQTHDMTIKKYREEYGEDVPLRPDDKA Microchaete P08041 MTPLMIRIRQEHRGIAEEVTQLFKDTQEFLSVTTAQ 65diplosiphon RQAQAKEQAENLHQFHKDLEKDTEEFLTDTAKERMAKAKQQAEDLFQFHKEMAENTQEFLSETAKER MAQAQEQARQLREFHQNLEQTTNEFLADTAKERMAQAQEQKQQLHQFRQDLFASIFGTF Nostoc sp. Q8YUS9MTALMVRIRQEHRSIAEEVTQLFRETHEFLSATTA 66HRQEQAKQQAQQLHQFHQNLEQTTHEFLTETTTQ RVAQAEAQANFLHKFHQNLEQTTQEFLAETAKNRTEQAKAQSQYLQQFRKDLFASIFGTF

TABLE 7Amino acid sequences of exemplary GVA proteins from B. megaterium. GVASEQ ID Protein Amino acid sequence NO.: gvpRMEIKKIMQAVNDFFGEHVAPPHKITSVEATEDEGWRVIVEVIEERE 67YMKKYAKDEMLGTYECFVNKEKEVISFKRLDVRYRSAIGIEA gvpNMTVLTDKRKKGSGAFIQDDETKEVLSRALSYLKSGYSIHFTGPAG 68GGKTSLARALAKKRKRPVMLMHGNHELNNKDLIGDFTGYTSKKVIDQYVRSVYKKDEQVSENWQDGRLLEAVKNGYTLIYDEFTRSKPATNNIFLSILEEGVLPLYGVKMTDPFVRVHPDFRVIFTSNPAEYAGVYDTQDALLDRLITMFIDYKDIDRETAILTEKTDVEEDEARTIVTLVANVRNRSGDENSSGLSLRASLMIATLATQQDIPIDGSDEDFQTLCIDILHHPLTKCLDEENAKSKAEKIILEECKNIDTEEK gvpFMSETNETGIYIFSAIQTDKDEEFGAVEVEGTKAETFLIRYKDAAMV 69AAEVPMKIYHPNRQNLLMHQNAVAAIMDKNDTVIPISFGNVFKSKEDVKVLLENLYPQFEKLFPAIKGKIEVGLKVIGKKEWLEKKVNENPELEKVSASVKGKSEAAGYYERIQLGGMAQKMFTSLQKEVKTDVFSPLEEAAEAAKANEPTGETMLLNASFLINREDEAKFDEKVNEAHENWKDKADFHYSGPWPAYNFVNIRLKVEEK gvpGMLHKLVTAPINLVVKIGEKVQEEADKQLYDLPTIQQKLIQLQMMF 70ELGEIPEEAFQEKEDELLMRYEIAKRREIEQWEELTQKRNEES gvpLMGELLYLYGLIPTKEAAAIEPFPSYKGFDGEHSLYPIAFDQVTAVV 71SKLDADTYSEKVIQEKMEQDMSWLQEKAFHHHETVAALYEEFTIIPLKFCTIYKGEESLQAAIEINKEKIENSLTLLQGNEEWNVKIYCDDTELKKGISETNESVKAKKQEISHLSPGRQFFEKKKIDQLIEKELELHKNKVCEEIHDKLKELSLYDSVKKNWSKDVTGAAEQMAWNSVFLLPSLQITKFVNEIEELQQRLENKGWKFEVTGPWPPYHFSSFA gvpSMSLKQSMENKDIALIDILDVILDKGVAIKGDLIISIAGVDLVYLDLR 72VLISSVETLVQAKEGNHKPITSEQFDKQKEELMDATGQPSKWTNP LGS gvpKMQPVSQANGRIHLDPDQAEQGLAQLVMTVIELLRQIVERHAMRR 73VEGGTLTDEQIENLGIALMNLEEKMDELKEVFGLDAEDLNIDLGP LGSLL gvpJMAVEHNMQSSTIVDVLEKILDKGVVIAGDITVGIADVELLTIKIRLI 74VASVDKAKEIGMDWWENDPYLSSKGANNKALEEENKMLHERLK TLEEKIETKR gypTMATETKLDNTQAENKENKNAENGSKEKNGSKASKTTSSGPIKRA 75VAGGIIGATIGYVSTPENRKSLLDRIDTDELKSKASDLGTKVKEKSKSSVASLKTSAGSLFKKDKDKSKDDEENVNSSSSETEDDNVQEYDELKEENQTLQDRLSQLEEKMNMLVELSLNKNQDEEAEDTDSDEEENDENDENDENEQDDENEEETSKPRKKDKKEAEEEESESDEDSEEEEEDSRSNKKNKKVKTEEEDEDESEEEKKEAKPKKSTAKKSKNTK AKKNTDEEDDEATSLSSEDDTTAgvpU MSTGPSFSTKDNTLEYFVKASNKHGFSLDISLNVNGAVISGTMISA 76KEYFDYLSETFEEGSEVAQALSEQFSLASEASESNGEAEAHFIHLKNTKIYCGDSKSTPSKGKIFWRGKIAEVDGFFLGKISDAKSTSKKSS

TABLE 8 Amino acid sequences of exemplary GVA proteins from Serratia sp.GVA SEQ ID Protein Amino acid sequence NO.: gvpNMIKQNTVSQYTVDDDLVVPEASEHFVATSYVNDIIERALVYLRAG 77YPVHFAGPSGIGKTTLAFHLAALWGRPVTMLQGNEEFVSSDLTGKDIGYRKSSLVDNYIHSVLKTEEQMNRMWVDNRLTTACRNGDMLIYDEFNRSKAETNNVLLSVLSEGILNLPGLRGVGEGYLDVHPEFRAIFTSNPEEYAGTHKTQDALMDRMITINIGLVDRDTELQILHARSELELKEAAYIVDIIRELRGNEHETKHGLRAGIAIAHILHQQGIKPRYGDKLFHAICYDVLSMDAAKIQHAGRSIYREMVDGVIRKICPPIGSDTVK ASTQKIKAVE GvpVMAISTRPLRTLSDIKTHSGRVSGEHQTYRDYFQIGALELERWRRTR 78EREAASSRIASIDERIADIDKEKAALLADATAASAVAENNDKSEAA EKKKKSSGLRIKY gvpF1MMSIDKSRNHRAKVLYALCVSDDSTPNYKIRGLEAAPVYSIDQDG 79LRAVVSDTLSTRLRPERRNITAHQAVLHKLTEEGTVLPMRFGVIARNAEAVKNLLVANQDTIREHFERLDGCVEMGLRVSWDVTNIYEYFVATYPVLSETRDEIWNGNSNANNHREEKIRLGNLYESLRSGDRKESTEKVKEVLLDYCEEIIENPVKKEKDVMNLACLVARERMDEFAKGVFEASKLFDNVYLFDYTGPWAPHNFVTLDLHAPTAKKKTLTRA GTLSD GvpF2MTMNTEAQTEQAIYLYGLTLPDLAAPPILGVDNQHPINTHQCAGL 80NAVISPVALSDFTGEKGEDNVQNVTWLTPRICRHAQIIDSLMAQGPVYPLPFGTLFSSQNALEQEMKSRATDVFVSLRRITGCQEWALEATLDRKQAVDVLFTEGLDSGRFCLPEAIGRRHLEEQKLRRRLTTELSDWLAHALTAMQNELHPLVRDFRSRRLLDDKILHWAYLLPVEDVAAFQQQVADIVERYEAYGFSFRVTGPWAAYSFCQPDES gvpF3MSLLLYGIVAEDTQLALEPDGSPHAGEEPMQLVKAATLAALVKPC 81EADVSREPAAALAFGQQIMHVHQQTTIIPIRYGCVLADEDAVTQHLLNHEAHYQTQLVELENCDEMGIRLSLASAEDNAVTTPQASGLDYLRSRKLAYAVPEHAERQAALLNNAFTGLYRRHCAEISMFNGQRTYLLSYLVPRTGLQAFRDQFNTLANNMTDIGVISGPWPPYNFAS gvpGMLLIDDILFSPVKGVMWIFRQIHELAEDELAGEADRIRESLTDLYM 82LLETGQITEDEFEQQEAVLLDRLDALDEEDDMLGDEPGDDEDDEYEEDDDEEDDDEEDDDDEDDDDEDDDDEEDDDDDEDDDDEDEPE GTTK GvpWMKPAIYPKFLLESPLKLVFFGGKGGVGKSTCATSTALRLAQEQPQ 83HHFLLVSTDPAHSLQNILSDLVLPKNLDVRELNAAASLHEFKSQHEGVLKEIAYRGTVLDQNDVQGLMDTALPGMDELAAYLEIAEWIQKDTYYRIIIDTAPTGHTLRLLEMPDLIYRWLTALDTLLAKQRYIRKRFAGDNRLDHLDHFLLDMNDSLKAMHELVTDSTRCCFVLVMLAEAMSVEESIDLAGALNQQRVFLSDLVVNRLFPENDCPTCCVERNRQMLALQNGYQRLPGHVFWTLPLLAIEPRGALLHEFWSGVRLLDENEVMATTCHHQLPLRVESSISLPASTFRLLIFAGKGGVGKTTLACATALRLNSEYPELRILLFSADPAHSLSDCLGVTLQQQPISVLVNIDAQEINAQADFDKIRQGYRAELEAFLLDTLPNLDITFDREVLEHLLDLAPPGLDEIMALTAIMDHLDSGRYDMVIVDGAPSGHLLRLLELPELIRDWLKQFFSLLLKYRKVMRFPHLSERLVQLSRELKNLRALLQDTKQTGLYAVTVPTHLALEKTYEMTCALQRLGLTANALFINQITPPSDCTLCQAITSRESLELKCADEMFPSQPHAQIFRQTEPTGLSKLKTLGSALF L gvpKMTTNQLSHHSPVFGPTSPAIQRPITEANRHKIDIDGERVRDGLAQL 84VLTLVKLLHELLERQAIRRMDSGSLSDEEVERLGLALMRQAEELT HLCDVFGFKDDDLNLDLGPLGRLLGvpX MVNTTNDINAATRGLLLRMGNAWFEQDELRQAVDIYLKIIEQYPD 85SKESKTAQTALLTISQRYERDGLFRLSLDILERVGEITPTSI gvpYMRALIHFPIIHSPKDLGTLSEAASHLRTETQTRAYLAAVEGFWTMI 86TTTIEGLDLDYTHLKLYQDGLPVCGKENEIVTDVANAGSQNYKLLLTLQHKGAILMGTESPELLLQERDLMTQLLQSTEQTEASLETAKTLLNRRDDYIAQRIDETLQDGEMAILFLGLMHNIEAKLPADIVFIQPL GKPPGGESI gvpHMTGNVEGILRGLGDLVEKLVETGEQIKRSGAFDIDTNDGKNAKAV 87YGFSIKMGLDGNQENRVEPFGNIRRDEQTGEATVQEVSEPLVDVIEESDHVLVLAEMPGVADEDVQVELNGDILTLHSERGSKKYHKEIVL PCSFDDKAMERSCRNGILEVKLGKGvpZ MSEELKLKVAEALPKDAGRGYARLDPADMARLNLAVGDIVQLTS 88KKGTGIAKLMPTYPDMRNKGIVQLDGLTRRNTSLSLDEKVQIEPASCKHATQIVLIPTTITPNQRDLDYIGSLLDGLPVQKGDLLRAHLFGSRSADFKVESTIPDGAVLIDPTTTLVIGKSNAVGNSSHSTQRLSYEDVGGLKNQVRRIREMIELPLRYPEVFERLGIDAPKGVLLSGPPGCGKTLIARIIAQETDAQFFTISGPEIVHKFYGESEAHLRKIFEEAGRKGPSIIFLDEIDSIAPHRDKVVGDVEKRIVAQLLALMDGLKNRGKVIVIAATNLPNAIDPALRRPGRFDREISIPIPDREGRREIIEIHSTGMPLNADVDLNVLADITHGFVGADLEALCREAAMSALRRLLPEIDFSSAELPYDRLAELTVMMDDFRAALCEVSPSAIRELFVDIPDVRWEDVGGLDDVRRRLIESVEWPIKYPELYEQAGVKPPKGLLLAGPPGVGKTLIAKAVANESGVNVISVKGPALMSRYVGDSEKGVRELFLKARQAAPCIIFLDEVDSVIPARNEGAIDSHVAERVLSQFLSEMDGLEELKGVFVMGATNRADLIDPAMLRPGRFDEIIELGLPDEDARRQILAVHLRNKPLGDNIHADDLAERCDGASGAELAAVCNRAALAALRRAIQQSEEAVLSPSTVGETPVALTVRIEQHDFAEVIAEMFGDDA

Example 10: Alignment of Exemplary gvpA and gvpB Protein Sequences

FIG. 18 shows an exemplary Clustal omega alignment of amino acidsequences of selected exemplary gvpA and gvpB proteins. The gvpA andgvpB proteins shown are from the following species: Sa_A2, Serratia sp.ATCC 39006 gvpA2; Sa_A3, Serratia sp. ATCC 39006 gvpA3; Sc_A2,Streptomyces coelicolor gvpA2; Sc_A1, Streptomyces coelicolor gvpA1;Fc_A, Frankia sp. gvpA; Bm_B1, B. megaterium gvpB1; Mb_A, Methanosarcinabarkeri gvpA; Hv_A, Halorubrum vacuolatum gvpA; Hm_A, Haloferaxmediterranei gvpA; Hs_A1, Halobacterium sp. NRC-1 gvpA1; Hs_A2,Halobacterium sp. NRC-1 gvpA2; Bm_A, B. megaterium gvpA; Bm_B2, B.megaterium gvpB2; Af_A, A. floc-aquae gvpA; Ma_A; Sa_A1, Serratia sp.ATCC 39006 gvpA1. The bottom row of FIG. 18 indicated as “Consensus”shows an exemplary consensus sequence derived from alignment of the gvpAand gvpB amino acid sequences shown.

In some embodiments described herein, homology-based searching (e.g.,BLAST alignment) of sequences of proteins encoded in the genome of aprokaryotic organism compared to the exemplary consensus sequence shownin FIG. 18 can be used to identify gvpA and/or gvpB protein sequences inthe prokaryotic organism.

Example 11: Exemplary GVGC Polynucleotide Construct to Allow Expressionof Two Different GV Types in One Cell

FIG. 19 shows an exemplary configuration of a construct designed toallow expression of two different GV types in one prokaryotic cell. Theexemplary construct in FIG. 19 is designed to provide alternatingexpression of two GV types, the first GV type encoded by Cluster 1, andthe second GV type encoded by Cluster 2, shown as block-shaped arrowsfacing in opposite orientations of a DNA strand (shown as a straightline), with a promoter between the two clusters. The promoter is flankedby recombination sites (e.g. flippase recognition target, FRT sites)shown as circles. For example, initially, the promoter can be orientedin a direction operatively linked to Cluster 1, initiating expression ofgyp genes for the formation of GV type 1. In presence of a cognaterecombinase (e.g. flippase, Flp), expressed from another geneticconstruct in the prokaryotic cell, the orientation of the promoter isreversed upon recombination at the FRT sites, and thereafter is orientedin the opposite direction, operatively linked to Cluster 2, initiatingexpression of gyp genes for the formation of GV type 2.

Example 12: Exemplary Gas Vesicle Gene Clusters

FIG. 20 shows diagrams illustrating the organization of exemplary gasvesicle gene clusters. Gas vesicle gene clusters from the indicatedorganisms are shown, with genes shown as block-shaped arrows, and genesof predicted similar function indicated in the same shade of grey. Thedirection of the transcription of genes within a gene cluster isindicated by the direction of the block-shaped arrows, and genes groupedtogether having block arrows pointed in the same direction are typicallyorganized in the same operon. The scale bar indicates 1 kb [1]. Inaddition, FIG. 21 shows diagrams illustrating organization of exemplarygyp gene clusters, wherein each letter indicates a gyp gene, and anarrow beneath a group of letters indicates an operon, with the directionof the arrow indicating the direction of transcription [2].

Example 13: Phylogenetic Relationships Between Exemplary gvpA, gvpF andgvpN Proteins

FIG. 22 shows exemplary phylogenetic relationships of the gvpA proteinsequences from the indicated prokaryotic species [1]. In someembodiments described herein, the identification of a GvpA/B protein canbe performed by comparing the sequence of an unknown protein in aprokaryotic cell with that of a known gvpA sequence from the closestphylogenetic relative of the prokaryotic species, such as thoseindicated in the exemplary phylogenetic tree diagram in FIG. 22.

FIG. 23 shows exemplary phylogenetic relationships of the gvpF and gvpLprotein sequences from the indicated prokaryotic species [1]. In someembodiments described herein, the identification of a GvpF protein canbe performed by comparing the sequence of an unknown protein in aprokaryotic cell with that of a known gvpF sequence from the closestphylogenetic relative of the prokaryotic species, such as thoseindicated in the exemplary phylogenetic tree diagram in FIG. 23.

FIG. 24 shows exemplary phylogenetic relationships of the gvpN proteinsequences from the indicated prokaryotic species [1]. In someembodiments described herein, the identification of a GvpN protein canbe performed by comparing the sequence of an unknown protein in aprokaryotic cell with that of a known gvpN sequence from the closestphylogenetic relative of the prokaryotic species, such as thoseindicated in the exemplary phylogenetic tree diagram in FIG. 24.

Example 14: Detection of GVs Production in Prokaryotic Cell

Detection of GVs in cells can be through determining if 1) the cellsbecome buoyant after centrifugation or 2) through detecting them usingtransmission electron microscopy. 3) cells can be lysed, the lysate canbe centrifuged for 4-16 hours at 300×g and the top 60 μl of solution canbe analyzed using Transmission electron microscopy.

After the cells have been expressing GV proteins, after 16-24 hours ofexpression, cells in a liquid culture can be put in 1-50 mL tubes andcentrifuged for 1-2 hours at 300×g.

After the cells have been expression GV proteins, after 16-24 hours ofexpression, cells from a liquid culture can be placed on TEM grids.Skilled person can look for hollow vesicles with the right size andshape indicating that GVs have been expressed and formed as per FIG. 2C.

Cells can be lysed using detergents provided in our manuscript. Thelysate can be centrifuged for 4-16 hours at 300×g. The top 60 μl of thesolution can be analyzed using TEM.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the hybrid GVGCs, and related GVR geneticcircuits, vectors, genetically engineered prokatyoric cells,compositions, methods and systems of the disclosure, and are notintended to limit the scope of what the inventors regard as theirdisclosure. Those skilled in the art will recognize how to adapt thefeatures of the exemplified hybrid GVGCs, and related genetic circuits,vectors, genetically engineered prokaryotic cells, compositions, methodsand systems herein disclosed to additional hybrid GVGCs, and relatedgenetic circuits, vectors, genetically engineered prokaryotic cells,compositions, methods and systems according to various embodiments andscope of the claims.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe disclosure pertains.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference. All referencescited in this disclosure are incorporated by reference to the sameextent as if each reference had been incorporated by reference in itsentirety individually. However, if any inconsistency arises between acited reference and the present disclosure, the present disclosure takesprecedence. Further, the computer readable form of the sequence listingof the ASCII text file P2075-US-Seq-List-ST25 is incorporated herein byreference in its entirety.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure claimed. Thus, it should be understood that although thedisclosure has been specifically disclosed by embodiments, exemplaryembodiments and optional features, modification and variation of theconcepts herein disclosed can be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise. The term “plurality” includestwo or more referents unless the content clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible sub-combinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the disclosure, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, systemelements, and materials other than those specifically exemplified may beemployed in the practice of the disclosure without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis disclosure. Whenever a range is given in the specification, forexample, a temperature range, a frequency range, a time range, or acomposition range, all intermediate ranges and all subranges, as wellas, all individual values included in the ranges given are intended tobe included in the disclosure. Any one or more individual members of arange or group disclosed herein may be excluded from a claim of thisdisclosure. The disclosure illustratively described herein suitably maybe practiced in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. Thespecific embodiments provided herein are examples of useful embodimentsof the disclosure and it will be apparent to one skilled in the art thatthe disclosure can be carried out using a large number of variations ofthe genetic circuits, genetic molecular components, and methods stepsset forth in the present description. As will be obvious to one of skillin the art, methods and systems useful for the present methods andsystems may include a large number of optional composition andprocessing elements and steps.

In particular, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

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The invention claimed is:
 1. A hybrid gas vesicle gene cluster (GVGC)encoding a hybrid gas vesicle (GV) type, the hybrid gas vesicle genecluster configured for expression in a prokaryotic host and comprisinggas vesicle assembly (GVA) genes comprising GvpR, GvpN, GvpF, GvpG,GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU native to Bacillus Megaterium, andone or more gas vesicle structural (GVS) genes comprising GvpA native toAnabaena flos-aquae, wherein the one or more gas vesicle structuralgenes and the gas vesicle assembly genes are in operative connection toallow co-expression in the prokaryotic host of the gas vesiclestructural genes and the gas vesicle assembly genes to provide thehybrid gas vesicle (GV) type in the prokaryotic host.
 2. The hybrid gasvesicle gene cluster (GVGC) of claim 1, wherein the prokaryotic host isBacillus Megaterium.
 3. The hybrid gas vesicle gene cluster (GVGC) ofclaim 1, wherein the prokaryotic host is a prokaryote of a prokaryoticspecies different from Bacillus Megaterium.
 4. The hybrid gas vesiclegene cluster (GVGC) of claim 1, wherein the prokaryotic host is E. coli,or Salmonella.
 5. The hybrid gas vesicle gene cluster (GVGC) of claim 1,wherein the GVS genes from Anabaena flos-aquae further comprises GvpC.6. The hybrid gas vesicle gene cluster (GVGC) of claim 1, wherein theGVS genes comprise a GVS gene encoding at least one GVS variantengineered to present a tag allowing clustering of expressedcorresponding GV types in the prokaryotic host.
 7. A vector comprisingthe hybrid GVGC of claim 1, configured for expression in a prokaryotichost, wherein the vector is configured to introduce the hybrid GVGC intothe prokaryotic host.
 8. A genetically engineered prokaryotic hostcomprising one or more hybrid GVGC of claim 1 configured for expressionin the genetically engineered prokaryotic host.
 9. The geneticallyengineered prokaryotic host of claim 8, wherein the prokaryotic host isE. Coli.
 10. The hybrid gas vesicle gene cluster (GVGC) of claim 1,wherein GVGC is ARG1 gene cluster having SEQ ID NO:
 16. 11. The hybridgas vesicle gene cluster (GVGC) of claim 1, wherein GVGC is ARG2 genecluster having SEQ ID NO:
 17. 12. A method to provide a hybrid gasvesicle gene cluster (GVGC) configured for expression in a prokaryotichost, the hybrid gas vesicle gene cluster encoding a gas vesicle (GV)type, the method comprising: providing a polynucleotidic constructcomprising gas vesicle assembly (GVA) genes and gas vesicle structural(GVS) genes forming a candidate GV gene cluster, the gas vesicleassembly (GVA) genes native to a GVA prokaryotic species and capable offorming detectable GVs in the prokaryotic host, and the gas vesiclestructural (GVS) genes native to one or more GVS prokaryotic species,with at least one of the one or more GVS prokaryotic species differentfrom the GVA prokaryotic species, providing a prokaryotic host of aprokaryotic species different from the GVA prokaryotic species;transforming the candidate GV gene cluster into the prokaryotic host;detecting expression in the prokaryotic host of one or more candidate GVgene cluster and repeating the detecting until formation of a GV by atleast one candidate GV gene cluster is detected, identifying thedetected at least one candidate GV gene cluster as the hybrid gasvesicle gene cluster (GVGC) configured for expression in the prokaryotichost.
 13. The method of claim 12, wherein the prokaryotic host is aGram-negative bacteria.
 14. The method of claim 13, wherein theprokaryotic host is E. coli, or Salmonella.
 15. The method of claim 12,wherein the prokaryotic host is Halobacterium.
 16. The method of claim12, wherein the gas vesicle assembly (GVA) genes forming the candidateGV gene cluster comprise GVA genes from Bacillus Megaterium Anabaenaflos-aquae Serratia sp., Bukholderia thailandensis, B. megaterium,Frankia sp, Haloferax mediaterranei, Halobacterium sp, Halorubrumvacuolatum, Microcystis aeruginosa, Methanosarcina barkeri, Streptomycescoelicolor, and/or Psychromonas ingrahamii.
 17. The method of claim 12,wherein the gas vesicle structural (GVS) genes forming the candidate GVgene cluster comprise GVS genes from Bacillus Megaterium Anabaenaflos-aquae Serratia sp., Bukholderia thailandensis, B. megaterium,Frankia sp, Haloferax mediaterranei, Halobacterium sp, Halorubrumvacuolatum, Microcystis aeruginosa, Methanosarcina barkeri, Streptomycescoelicolor, and/or Psychromonas ingrahamii.
 18. The method of claim 12,wherein the gas vesicle assembly (GVA) genes comprise GVA genes fromBacillus Megaterium, Anabaena flos-aquae, and/or Serratia sp., and thegas vesicle structural (GVS) genes forming the candidate GV gene clustercomprise GVS genes from Bacillus Megaterium Anabaena flos-aquaeBukholderia thailandensis, B. megaterium, Frankia sp, Haloferaxmediaterranei, Halobacterium sp, Halorubrum vacuolatum, Microcystisaeruginosa, Methanosarcina barkeri, Streptomyces coelicolor, and/orPsychromonas ingrahamii.
 19. The method of claim 12, wherein the gasvesicle assembly (GVA) genes forming the candidate GV gene clustercomprise GVA genes from Bacillus Megaterium, Anabaena flos-aquae, and/orSerratia sp., and the gas vesicle structural (GVS) genes forming thecandidate GV gene cluster comprise GVA genes from Bacillus Megateriumand/or Anabaena flos-aquae.
 20. The method of claim 12, wherein the gasvesicle assembly (GVA) genes comprise B. megaterium GVA genes GvpR,GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and/or GvpU.
 21. Themethod of claim 12, wherein the gas vesicle assembly (GVA) genescomprise B. megaterium GVA genes GvpA, GvpF, GvpG, GvpJ, GvpL, GvpK,GvpS, GvpU.
 22. A method to produce a hybrid gas vesicle type in aprokaryotic host, the method comprising: introducing into theprokaryotic host a hybrid gas vesicle gene cluster (GVGC), encoding ahybrid gas vesicle (GV) type, the hybrid gas vesicle gene clusterconfigured for expression in a prokaryotic host and comprising gasvesicle assembly (GVA) genes comprising GvpR, GvpN, GvpF, GvpG, GvpL,GvpS, GvpK, GvpJ, GvpT and GvpU native to Bacillus Megaterium, and oneor more gas vesicle structural (GVS) genes comprising GvpA native toAnabaena flos-aquae, wherein the one or more gas vesicle structuralgenes and the gas vesicle assembly genes are in operative connection,the introducing performed under condition allowing co-expression of thehybrid GVGC in the prokaryotic host of the gas vesicle structural genesand the gas vesicle assembly genes to produce the gas vesicle type inthe prokaryotic host.
 23. The method of claim 22, wherein theprokaryotic host is a Gram-negative bacteria.
 24. The method of claim23, wherein the prokaryotic host is E. coli, or Salmonella.
 25. Themethod of claim 22, wherein the prokaryotic host is Halobacterium.
 26. Ahybrid gas vesicle obtained by the method of claim
 22. 27. A method toimage a target site comprising a prokaryotic host, the methodcomprising: introducing into the prokaryotic host a hybrid gas vesiclegene cluster (GVGC) encoding a hybrid gas vesicle (GV) type, the hybridgas vesicle gene cluster configured for expression in a prokaryotic hostand comprising gas vesicle assembly (GVA) genes comprising GvpR, GvpN,GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU native to BacillusMegaterium,and one or more gas vesicle structural (GVS) genes comprisingGvpA native to Anabaena flos-aquae, wherein the one or more gas vesiclestructural genes and the gas vesicle assembly genes are in operativeconnection to allow co-expression in the prokaryotic host of the gasvesicle structural genes and the gas vesicle assembly genes to producethe hybrid gas vesicle type in the prokaryotic host, and imaging thetarget site comprising the prokaryotic host by applying a magnetic fieldand/or ultrasound to obtain an MRI and/or an ultrasound image of thetarget site.
 28. The method of claim 27, wherein the prokaryotic host isa Gram-negative bacteria.
 29. The method of claim 28, wherein theprokaryotic host is E. coli, or Salmonella.
 30. The method of claim 27,wherein the prokaryotic host is Halobacterium.